Recombinant mumps virus vaccine

ABSTRACT

The present invention provides the complete genomic sequence of the epidemic mumps virus (MuV) strain MuV Iowa/us/06 . Further, a reverse genetics system was constructed and used to rescue recombinant viral constructs that are attenuated compared to MuV Iowa/us/06  and JL vaccine viruses. Such constructs include viral constructs lacking the open reading frame (ORF) of the SH gene (rMuVΔSH) and/or incapable of expressing the V protein (rMuVΔV).

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 61/446,619, filed Feb. 25, 2011, and U.S. Provisional ApplicationSer. No. 61/529,981, filed Sep. 1, 2011, each of which is incorporatedby reference herein.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.K02AI065795, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND

Mumps virus (MuV), a paramyxovirus, causes acute parotitis in humans,characterized by lateral or bilateral swelling of the salivary glands.MuV is also notable as a highly neurotropic and neurovirulent agentcausing a number of central nervous system (CNS) manifestations rangingfrom mild meningitis to severe, and occasionally fatal, encephalitis.Mumps virus infection was the most common cause of viral meningitis andencephalitis until the arrival of mass immunization with mumps virusvaccine. The incidence of mumps and its complications were dramaticallyreduced following the introduction of measles, mumps, rubella vaccine(MMR) in 1971. MMR vaccine containing the Jeryl Lynn (JL) strain, anattenuated strain of MuV, is highly efficacious and produces few adversereactions. Currently, mumps virus vaccination is a part of a two doseMMR (mumps, measles, and rubella) vaccine regimen that is administratedto children at one and five years of age in the United States.

In recent years, MuV has caused epidemics among highly vaccinatedpopulations. In 2006, the U.S. experienced the largest mumps epidemic innearly 20 years (Marin et al., 2008, Vaccine; 26(29-30):3601-3607). Theoutbreak originated at a university in Iowa and spread to eleven otherstates. Over 5000 mumps cases were reported in 2006 compared to anaverage of approximately 250 cases/year in the previous decade. In2009-2010, a mumps outbreak occurred in the State of New York and theState of New Jersey in the US in which 88% of the patients had one-doseof mumps vaccine and 75% of the patients had two doses of vaccine (MMWRMorb Mortal Wkly Rep; 59(5):125-129, 2010).

While definitive causes for these recent outbreaks are not known,possible reasons (not mutually exclusive) for these outbreaks includewaning immunity, high velocity of infection, and vaccine failure due toemerging of a new mumps virus strain. See, for example, (Crowley andAfzal, 2002, Commun Dis Public Health; 5(4):311-313; Lim et al., 2003, JMed Virol; 70(2):287-292; Otto et al., 2010, Euro Surveill; 15(50);Strohle et al., 1996, Arch Virol; 141(3-4):733-741; Utz et al., 2004, JMed Virol; 73(1):91-96; and Whelan et al., 2010, Euro Surveill; 15(17).The results of a large study to examine the efficacy of the two-dose MMRagainst mumps virus by CDC indicate that titers of anti-MuV droppeddramatically 12 years after the second dose of MMR (17 years of age), tothe level of pre-second dosage inoculation. Furthermore, neutralizingantibody titers are low in adults: out of 101 sera tested, 74 werepositive using ELISA and only one had neutralization antibody titerhigher than 1:8. This is consistent with the fact that in the 2006outbreak, the most affected population was 18 to 24 years of age. In the2010 outbreak, most affected patients were 13 to 14 years of age. Bothrecent outbreaks occurred in high-density populations (college campusand religious school). High velocity infection (for example, largequantity of infectious virions transmitted from one to another due toclose contact) may have overwhelmed the anti-MuV immunity in recentoutbreaks.

The current vaccine Jeryl Lynn (JL) is based in MuV genotype A, whilerecent outbreaks have been caused by genotype G. It is possible thatvaccine generated immunity based on strain A is ineffective inpreventing infection of strain G, leading to the outbreak. Because ofre-emerging of mumps virus outbreaks even in vaccinated populations,mumps virus has been listed as a high priority pathogen by NationalInstitute of Allergy and Infectious Diseases (see “Emerging andRe-emerging Infectious Diseases” on the worldwide web atniaid.nih.gov/topics/emerging/list.htm). Currently, live attenuated MuVvaccines are obtained through serial passages in embryonic eggs andcells. This is a time consuming process and a strategy with a poorrecord of generating safe vaccines.

Thus, there is a need for new and improved mumps vaccines, including thedevelopment of vaccines directed at the genotype G and a need for newand improved methods for developing mumps vaccines.

SUMMARY OF THE INVENTION

The present invention includes an isolated nucleotide sequence includinga cDNA sequence encoding the full length RNA genome of a mumps virus,wherein the isolated nucleotide sequence encodes a mumps virus unable toexpress a small hydrophobic (SH) protein product and/or unable toexpress a V protein product, and fragments and derivatives thereof.

In some aspects, an isolated nucleotide sequence including a cDNAsequence encoding the full length RNA genome of a mumps virus unable toexpress a small hydrophobic (SH) protein product includes a deletion ofthe open reading frame (ORF) encoding the SH protein, a mutationconverting a start codon into a stop codon, or a mutation in the regionbetween F protein ORF and the SH protein ORF that disrupts transcriptionof the SH gene. In some aspects, a deletion of the open reading frame(ORF) encoding the SH protein includes a deletion of 156 nucleotides ofthe ORF encoding the SH protein.

In some aspects, an isolated nucleotide sequence including a cDNAsequence encoding the full length RNA genome of a mumps virus unable toexpress a V protein product includes one or more mutations to the V/I/Pgene abrogating expression of the V protein. In some aspects, one ormore mutations to the V/I/P gene abrogating expression of the V proteininclude the nucleotide sequence GAGGAGGG at the editing site in the P/Vgene.

In some aspects, an isolated nucleotide sequence including a cDNAsequence encoding the full length RNA genome of a mumps virus includes adeletion of the open reading frame (ORF) encoding the SH protein or amutation converting a start codon into a stop codon and includes one ormore mutations to the V/I/P gene abrogating expression of the V protein.In some aspects, the one or more mutations to the V/I/P gene abrogatingexpression of the V protein include the nucleotide sequence GAGGAGGG atthe editing site in the P/V gene.

The present invention also includes an isolated nucleotide sequenceincluding a cDNA sequence encoding the full length RNA genome of a mumpsvirus as described herein, including one or more further mutationsand/or deletions. In some aspects, a further mutation or deletion mayinclude a mutation or deletion effecting phosphorylation of the Pprotein. In some aspects, a further mutation or deletion effectingphosphorylation of the P protein may include a mutation or deletion atT147 and/or S307 of the P protein.

The present invention also includes an isolated nucleotide sequenceincluding a cDNA sequence encoding the full length RNA genome of a mumpsvirus as described herein, further including expression of an I proteinproduct and/or further including mutations in the L protein product.

The present invention also includes an isolated nucleotide sequenceincluding a cDNA sequence encoding the full length RNA genome of a mumpsvirus as described herein, wherein the mumps genome further encodes aheterologous polypeptide.

In some aspects, an isolated nucleotide sequence including a cDNAsequence encoding the full length RNA genome of a mumps virus belongs togenotype G.

In some aspects, an isolated nucleotide sequence including a cDNAsequence encoding the full length RNA genome of a mumps virus isMuV/IowaUS/2006 (MuV-IA). In some aspects, MuV/IowaUS/2006 (MuV-IA)includes SEQ ID NO:1.

The present invention includes an isolated nucleotide sequence includinga cDNA sequence encoding the full length RNA genome of theMUV/IowaUS/2006 (MuV-IA) strain of the mumps virus, and fragments andderivatives thereof. In some aspects, the nucleotide sequence includesSEQ ID NO:1.

The present invention includes a recombinant mumps virus (rMuV) havingan isolated nucleotide acid sequence including a cDNA sequence encodinga full length RNA genome of a mumps virus, as described herein, or afragment or derivative thereof.

The present invention includes a plasmid encoding a measles virus genome(pMuV) including a cDNA sequence encoding a full length RNA genome of amumps virus, as described herein, or a fragment or derivative thereof.

The present invention includes a viral expression vector including anisolated nucleotide sequence including a cDNA sequence encoding the fulllength RNA genome of a mumps virus as described herein, or a fragment orderivative thereof.

The present invention includes an infectious viral particle including anisolated nucleotide sequence or plasmid as described herein.

The present invention includes a composition including an isolatednucleotide sequence, plasmid, pMuV, rMuV, or infectious viral particleas described herein. In some embodiments, a composition further includesa rubella and/or measles antigenic determinant. In some embodiments, thecomposition is formulated for intranasal, oral, intradermal, orintramuscular administration.

The present invention includes a method of inducing an immune responseto mumps virus in a subject, the method including administering aneffective amount of an isolated nucleotide sequence plasmid, pMuV, rMuV,viral particle, or composition as described herein, to the subject. Insome embodiments, administration includes intranasal, oral, intradermal,or intramuscular administration.

The present invention includes a method of vaccinating a subject againstmumps, the method including administering an effective amount of anisolated nucleotide sequence plasmid, pMuV, rMuV, viral particle, orcomposition as described herein to the subject. In some embodiments,administration includes intranasal, oral, or intramuscularadministration.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Analysis of MuV-IA genome. FIG. 1A is an alignment of theSH proteins. SH protein sequences of three strains, Glouc1/UK96 (SEQ IDNO:4), UK01-22 (SEQ ID NO:5) and MuV-IA (SEQ ID NO:6) are shown. MuV-IAwas different from Glouc1/UK96* and UK01-22 for only five nucleotidesand two amino acids respectively. The transmembrane domain of mumpsvirus SH protein, outlined in rectangular box, was predicted usingTMHMMserver v.2.0 (CBS; Denmark). FIG. 1B is a sequence comparison ofdifferent mumps viruses. The genome sequences of 32 strains of mumpsviruses were obtained from NCBI Genbank and aligned with genome sequenceof MuV-IA using MEGA4 version 4.0.2. The 32 MuV Strains were (accessionnumbers are given in the brackets): 87-1004 (AF314560), SIPAR 02(AF314558), Biken (AF314561), 87-1005 (AF314562), MuV (2001) (AF314559),Urabe 1004-10/2 (FJ375177), Urabe Gw7 (FJ375178), Hoshino (AB470486),Miyahara (1992) (NC_(—)002200), MuV Miyahara (1992) (2) (AB040874),Y213(AB576764), Dg1062/Korea/98 (32172464), L3/Russia/Vector (AY508995),L-Zagreb master seed (AY685921), L-Zagreb vaccine strain (AY685920),9218/Zg98 (299766355), Novosbrisk genotype C (50404164), PetroNovgenotype H (AY681495), 88-1961 (AF467767), Glouc1/UK96(AF280799),Du/CRO05 (EU370207), SP-A (FJ556896), SP (EU884413), SP (2006)(DQ649478), JL2 (AF3452901), Jeryl Lynn sub strain (FN31985), Enders(GU9800521), Jeryl Lynn major component (AF338106), MuV (2000)(AF201473), JL1 (FJ211586), RIT4385 (FJ211585), RIT4385(2) (FJ211584).FIG. 1C is a sequence comparison between MuV-IA and Jeryl Lynn (JL)strain (major component). The gene and protein sequences of NP, P/V(encoding P protein and V protein), M, F, SH, HN and L of MuV-IA andJeryl Lynn live vaccine major component were aligned using NCBI BLASTprogram. Nucleotide identities and amino acid identities were shownabove. *The SH genes were aligned using MEGA 4.0.2.

FIGS. 2A-2D. Analysis of rMuV, rMuV-EGFP and rMuV-RL. FIG. 2A shows thegrowth rate of rMuV. rMuV was obtained by transfecting BSRT-7 cells withpMuV-IA, pCAGGS-NP, pCAGGS-P and pCAGGS-L. Growth rates of rMuV (emptysquare) and MuV-IA (filled triangle) were compared. FIG. 2B shows viralprotein expression levels of rMuV and MuV-IA. Six well plates of Verocell were infected with mock, rMuV or MuV-IA at a MOI of 0.5. Celllysates were subjected to immunoblotting with anti-NP, P or V. FIG. 2Cshows rescue of rMuV-EGFP. rMuV-EGFP was rescued in a similar manner asdescribed for rMuV. Vero cell in six well plates were infected with mockor rMuV-EGFP at a MOI of 0.05 and photographed at 2 dpi. FIG. 2D showsrescue of rMuV-RL. rMuV-RL was recovered from cloned DNA as describedfor rMuV. 24 well plates of Vero cell were infected with mock or rMuV-RLat MOI of 0.1. At 2 dpi, cells were assayed for renilla luciferaseactivity.

FIGS. 3A-3D. Generation of a MuV lacking SH (rMuVΔSH). FIG. 3A is aschematic of the production of rMuVΔSH. The SH ORF (SEQ ID NO:7) wasreplaced with a 5 amino acid coding sequence containing an Nhe I site(SEQ ID NO:8; restriction site is underlined). FIG. 3B is confirmationof rMuVΔSH by RT-PCR. After plaque purification, viral RNA was extractedand was subjected to RT-PCR, in which two primers flanking the SH genewere used to perform a PCR to confirm the deletion. Lane 1 and Lane 6are 100 bp and 1 kb DNA ladder respectively; Lane 2 is the negativecontrol—PCR without polymerase. Lane 3, 4 and 5 are PCR products fromrMuVΔSH, rMuV and wtMuV-infected cells respectively. FIG. 3C isconfirmation of rMuVΔSH by sequencing. PCR products were sequenced (SEQID NO:9). The inserted sequence is underlined. FIG. 3D shows expressionof SH in MuV-IA, rMuV and rMuVΔSH-infected cells. Vero cells were mockinfected or infected with MuV-IA, rMuV or rMuVΔSH. Cell lysates weresubjected to immunoblotting with anti-NP, P or SH.

FIGS. 4A-4D. Growth rates and viral protein expression of rMuVΔSH andrMuV. FIG. 4A shows growth rates of rMuVΔSH and rMuV. Vero cell wereinfected with rMuVΔSH (filled diamond) and rMuV (empty square) at MOI of0.01. Supernatants were used for plaque assay. FIG. 4B shows viralprotein expression levels in rMuVΔSH and rMuV infected cells. Vero cellswere mock infected or infected with rMuVΔSH or rMuV at a MOI of 0.5.FIG. 4C shows HN expression level in rMuVΔSH and rMuV infected cells.Vero cells were mock infected or infected with rMuVΔSH or rMuV at a MOIof 0.5. Cells were collected at 24 hpi and expression levels of total HNand NP were examined using flow cytometry. Mean fluorescence intensity(MFI) of HN and NP were calculated. Y-axis represents the relative ratioof MFI of HN normalized by MFI of NP. FIG. 4D shows HN mRNA level inrMuVΔSH or rMuV infected Vero cells. Viral RNA was extracted fromrMuVΔSH or rMuV-infected Vero cells, reverse transcribed with oligodTand used for real time PCR. HN and F mRNA levels were calculated and RNmRNA level was normalized by F mRNA level. Y-axis represents ratio of HNmRNA verses F mRNA.

FIGS. 5A-5C. Induction of cell death by rMuVΔSH. FIG. 5A showscytopathic effects of rMuVΔSH or rMuV in tissue culture cell lines. Verocells, MDBK cells or HeLa cells were mock infected or infected withrMuVΔSH or rMuV at MOI of 0.01 and photographed at 1 dpi. FIG. 5B showscytopathic effects of rMuVΔSH infection in L929 cells. L929 cells weremock infected, or infected with rMuVΔSH, or rMuV at a MOI of 3 and werephotographed at 1 dpi and 2 dpi. FIG. 5C shows rMuVΔSH infection inducedapoptosis in L929 cells. L929 cells were infected as in FIG. 5B. At 1dpi, both floating cells and attached cells were collected, fixed,permeabilized and used for TUNEL assay.

FIGS. 6A-6D. The role of TNF-α in rMuVΔSH-mediated cell death. FIG. 6Ashows rMuVΔSH infection activated P65 in L929 cells. L929 cells on glasscover slips were infected with mock, rMuVΔSH or rMuV at a MOI of 10. At1 dpi, L929 cells on the cover slips were stained with anti-P65. FIG. 6Bshows rMuVΔSH infection in L929 cells induced TNF-α production. L929cells were mock infected or infected with rMuVΔSH, or rMuV at a MOI of5. TNF-α in the media was measured using ELISA. FIG. 6C shows treatmentwith anti-TNF-α antibody reduced CPE in rMuVΔSH infected L929 cells.L929 cells were mock infected or infected with rMuVΔSH or rMuV at a MOIof 5. Cells were cultured in media containing TNF-α neutralizingantibody or control antibody and were photographed at 1 dpi. FIG. 6Dshows TNF-α antibody treatment inhibited apoptosis in rMuVΔSH infectedL929 cells. L929 cells in 6 well plates were infected and treated as inFIG. 6C. At one day and two days post infection, cells were collectedand used for TUNEL assay.

FIGS. 7A and 7B. MuV-IA SH inhibited TNF-α activation of NF-κB. L929cells were transfected with a reporter plasmid (pKB-TATA-FL) andpCAGGS-MuV SH, pCAGGS-PIV5 SH or pCAGGSMuV-NP (FIG. 7A). At one daypost-transfection, cells were treated with TNF-α at 10 ng/ml for fourhours and then assayed for fire fly luciferase activity. Similarly, theeffect of the sequence of the SH ORF was examined using a plasmidencoding the SH ORF sequence without expressing the SH polypeptide dueto in-frame stop codon insertion downstream of the start codon of the SHORF (FIG. 7B).

FIG. 8. Neurotoxicity of MuVΔSH in vivo. Newborn rats were infectedintracerebrally with rMuV or rMuVΔSH. The animals were sacrificed at 30days post infection. The brains of the animals were sectioned, stainedand neurotoxicity scores were calculated based on relative hydrocephalusscore as described in the Material and Methods.

FIGS. 9A-9D. Generation of a MuV^(Iowa/US/06) lacking V protein(rMuV^(Iowa/US/06)ΔV). FIG. 9A is a schematic of rMuV^(Iowa/US/06)ΔV.The GGGGGG (nucleotides 1-6 of SEQ ID NO:14) editing site in the P/Vgene of MuV^(Iowa/US/06) was changed to GAGGAGGG (nucleotides 1-8 of SEQID NO:15) to eliminate expression of the V protein. To maintain thegenome length of rMuV^(Iowa/US/06)ΔV to be a multiple of six, fourbasepairs (bp) were added to the P/V gene 3′ UTR (SEQ ID NO:16). FIG. 9Bshows confirmation of the rescue of rMuV^(Iowa/US/06)ΔV. Viral RNAsextracted from rMuV^(Iowa/US/06)ΔV- and rMuV^(Iowa/US/06)-infected cellswere reverse transcribed into cDNA, followed by reverse transcription(RT)-PCR using two primers flanking the P/V gene. Lane 1 is 100-bp DNAladder; lane 2 is negative control (PCR without polymerase); lanes 3 and4 are PCR products from rMuV^(Iowa/US/06)ΔV- andrMuV^(Iowa/US/06)-infected cells, respectively. FIG. 9C is confirmationof rMuV^(Iowa/US/06)ΔV. The PCR products shown in FIG. 9B were sequenced(SEQ ID NO:17). FIG. 9D shows expression of the V protein inrMuV^(Iowa/US/06)ΔV- and rMuV^(Iowa/US/06)-infected cells. Vero cellswere mock infected or infected with rMuV^(Iowa/US/06)ΔV orrMuV^(Iowa/US/06). Cell lysates were immunoblotted using anti-NP, -P, or-V.

FIGS. 10A and 10B. Whole-genome sequencing of rescuedrMuV^(Iowa/US/06)ΔV. FIG. 10A is a summary of changes found in rescuedrMuV^(Iowa/US/06)ΔV. The leftmost panel shows the names of individualrMuV^(Iowa/US/06)ΔV strains from eight successful virus rescues. FIG.10B is a schematic of the changes that occurred in the NP GE and P/V GSregions. Changes that occurred during virus rescue ofrMuV^(Iowa/US/06)ΔV are indicated as bold, italic letters. The NP GE andP/V GS sequence in the plasmid is shown (SEQ ID NO:18). The NP GE andP/V GS mutated sequences are shown for the following virus strains:PX2-SP-48 (SEQ ID NO:19), PX2-sp-51 (SEQ II) NO:20), PX2-sp-61 (SEQ IDNO:21), PX2-sp-81 (SEQ ID NO:22), PX2-sp-91 (SEQ ID NO:23), PX2-sp-101(SEQ ID NO:24), and PX2-sp-106 (SEQ ID NO:25). The “TT” in the middle ofthe sequence alignment is the gene junction sequence between the NP andP/V genes in MuV^(Iowa/US/06).

FIGS. 11A-11E. Growth rates and viral protein expression ofrMuV^(Iowa/US/06)ΔV, rMuV^(Iowa/US/06), and MuV^(Iowa/US/06). FIG. 11Ashows growth rates of rMuV^(Iowa/US/06)ΔV and rMuV^(Iowa/US/06) in Verocells. Vero cells were mock infected or infected withrMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of 0.01. Supernatantswere collected for plaque assay. FIG. 11B shows plaques ofrMuV^(Iowa/US/06)ΔV and rMuV^(Iowa/US/06) in Vero cells.rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) was plated onto Vero cells. Theplaques were stained with Giemsa at 6 dpi. FIG. 11C shows viral proteinexpression of rMuV^(Iowa/US/06)ΔV and rMuV^(Iowa/US/06) in Vero cells.Vero cells were infected as in FIG. 11A. Viral protein levels wereexamined by immunoblotting with anti-NP and P. β-Actin was used as aloading control. FIG. 11D presents growth rates of rMuV^(Iowa/US/06)ΔVand rMuV^(Iowa/US/06) in HeLa cells. HeLa cells were infected as FIG.11A. FIG. 11E presents growth rates of rMuV^(Iowa/US/06)ΔV andrMuV^(Iowa/US/06) in 293T cells. 293T cells were mock infected orinfected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of 0.5.Supernatants were collected for plaque assay.

FIGS. 12A-12D. Ratios of NP and P in rMuV^(Iowa/US/06)ΔV-infected cells.FIG. 12A shows NP and P expression levels in rMuV^(Iowa/US/06)ΔV- andrMuV^(Iowa/US/06)-infected Vero cells during early time pointspostinfection. Vero cells were mock infected or infected withrMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of 0.5. Vero cellswere collected and examined for NP and P expression using flowcytometry. Ratios of mean fluorescence intensity (MFI) of P over NP areshown. P values of rMuV^(Iowa/US/06) versus rMuV^(Iowa/US/06)ΔV at 12and 16 hpi were calculated using Student's t test and are less than0.05. FIG. 12B shows NP and P expression levels in rMuV^(Iowa/US/06)ΔV-and rMuV^(Iowa/US/06)-infected Vero cells during late time pointspostinfection. Ratios of MFI of P over NP at multiple time pointspostinfection were examined as in FIG. 12A. P values ofrMuV^(Iowa/US/06) versus rMuV^(Iowa/US/06)ΔV at 24 and 48 hpi are lessthan 0.05. FIG. 12C shows NP and P expression ratios ofrMuV^(Iowa/US/06)ΔV (P GS). Ratios of MFI of P over NP inrMuV^(Iowa/US/06)ΔV (P GS)-infected Vero cells, at an MOI of 0.5, wereexamined at 24 hpi. P values of rMuV^(Iowa/US/06) versusrMuV^(Iowa/US/06)ΔV (P GS) are less than 0.05. FIG. 12D shows NP and Pexpression ratio of rMuV^(Iowa/US/06) (L gene). Ratios of MFI of P overNP in rMuV^(Iowa/US/06)ΔV (L gene)-infected Vero cells, at an MOI of0.5, were examined at 24 hpi. P values of rMuV^(Iowa/US/06) versusrMuV^(Iowa/US/06)ΔV (L gene) are less than 0.05.

FIG. 13. HN expression level in rMuV^(Iowa/US/06)ΔV. Vero cells weremock infected or infected with rMuVΔV or rMuV at an MOI of 0.5. Cellswere collected at 24 hpi and stained for BIN using flow cytometry.

FIGS. 14A-14C. Induction of cell death by rMuV^(Iowa/US/06)ΔV. In FIG.14A, rMuV^(Iowa/US/06)ΔV induced a greater cytopathic effect in celllines. HeLa cells, MDBK cells, or Vero cells were mock infected orinfected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of 0.5and photographed at 72 hpi. FIG. 14B shows induction of apoptosis byrMuVΔV in HeLa cells. HeLa cells were infected as in FIG. 14A. At 24hpi, cells were collected for TUNEL assay. Percentages of TUNEL-positivecells out of total cells are shown. The P value of rMuV^(Iowa/US/06)versus rMuV^(Iowa/US/06)ΔV is less than 0.05. FIG. 14C shows inductionof apoptosis in Vero cells. The Vero cells were infected with 0.5 MOI ofviruses and processed for TUNEL assay as in FIG. 14B. P values betweenthe wild type and rMuV^(Iowa/US/06)ΔV are less than 0.05.

FIGS. 15A and 15B. Degradation of STAT1 and STAT3 inMuV^(Iowa/US/06)-infected cells. As shown in FIG. 15A,rMuV^(Iowa/US/06)ΔV failed to degrade STAT1 in Vero cells. Vero cellswere infected at an MOI of 0.5. Cell lysates were immunoblotted withanti-NP, -P, -V, and anti-STAT1 recognizing both STAT1 isoforms (STAT1αand STAT1β). β-Actin was used as a loading control. As shown in FIG.15B, rMuV^(Iowa/US/06)ΔV failed to degrade STAT3 in Vero cells. Celllysates were immunoblotted with anti-NP, -P, -V, -STAT3, and -STAT2.

FIGS. 16A and 16B. Induction of IFN-β and IL-6 by rMuV^(Iowa/US/06)ΔV.FIG. 16A shows induction of IFN-β production by rMuV^(Iowa/US/06)ΔVvirus. 293T cells were infected with wild-type PIV5, rPIV5V C,rMuV^(Iowa/US/06), or rMuV^(Iowa/US/06)ΔV or mock infected. The cellularsupernatants were collected at 24 and 48 hpi and analyzed for IFN-βproduction by ELISA. The graph shows the average of three independentexperiments, and error bars represent the standard deviation (SD). Pvalues of rMuV^(Iowa/US/06) versus rMuV^(Iowa/US/06)ΔV at 24 and 48 hpiare less than 0.05. FIG. 16B shows induction of IL-6 production byrMuV^(Iowa/US/06)ΔV virus. HeLa cells were infected with wild-type PIV5,rPIV5V C, rMuV^(Iowa/US/06), or rMuV^(Iowa/US/06)ΔV or mock infected.The cellular supernatants were collected at 24 and 48 hpi and analyzedfor IL-6 production by ELISA. The test samples were diluted to 1:10 in asample diluent provided in the kit. The graph shows the average of twoindependent experiments, and error bars represent the SD. P values ofrMuV^(Iowa/US/06) versus rMuV^(Iowa/US/06)ΔV at 24 and 48 hpi are lessthan 0.05.

FIG. 17. Neurotoxicity of rMuV^(Iowa/US/06)ΔV in vivo. The severity ofhydrocephalus in rats inoculated with rMuV^(Iowa/US/06) orrMuV^(Iowa/US/06)ΔV was measured as described in Example 2. rMuVΔV-1 isrMuV^(Iowa/US/06)ΔV rescue #4 (PX2-SP-48) and rMuVΔV-2 isrMuV^(Iowa/US/06)ΔV rescue #5 (PX2-SP-51); as detailed in FIG. 10). Pvalues of rMuV versus rMuVΔV-1 or rMuVΔV-2 are less than 0.05. The Pvalue of rJL versus rMuVΔV-1 is less than 0.05. n=36 for MuV, n=16 forrMuVΔV-1, and n=18 for rMuVΔV-2.

FIGS. 18A and 18B. Titers of anti-MuV antibodies in the sera measuredusing ELISA. FIG. 18A shows titers measured at serial dilutions of thesera. FIG. 18B shows titers at a dilution of 1:1024.

FIG. 19. Schematics of MuV-F and F-MuV RSV F can be inserted between Fand SH to give rise to MuV-F or between leader sequence and NP to giverise to F-MuV. The insert is a more detailed diagram of F insertion.Sequences of gene start (GS), intergenic region (I) and gene end (GE),which are important for initiation and termination of viral mRNAsynthesis, are indicated.

DETAILED DESCRIPTION

In 2006, the U.S. experienced the largest mumps epidemic in nearly 20years (Marin et al., 2008, Vaccine; 26(29-30):3601-3607). The outbreakoriginated at a university in Iowa and spread to eleven other states.With the present invention, the sequence of the complete genome of aclinical wild-type isolate from the Iowa mumps epidemic has beendetermined. This isolate, the Iowa strain, also referred to herein asMuV-IA, rMuV^(Iowa/US/06), MuV Iowa/US/06, MuV-Iowa/US/06, or MuV(Iowa/US/06) is a member of genotype G, not genotype A of the widelyused Jeryl Lynn (JL) mumps vaccine. A reverse genetics system wasgenerated for this mumps virus, and using this reverse genetics system,various recombinant MuV constructs were generated, including, but notlimited to, recombinant MuV lacking the expression of the viral proteinsSH (rMuVΔSH) and/or V (rMuVΔV). These recombinant viruses grow well intissue culture cells such as Vero cells, which are WHO-approved cellline for vaccine production, but are attenuated in an animal model,demonstrating lower neurotoxicity than even the JL vaccine. Theserecombinant viruses and their derivatives are suitable for a newgeneration of MuV vaccines.

Mumps virus (MuV), a member of the family Paramyxoviridae, is a negativestranded, non-segmented RNA virus with a genome of 15,384 nucleotides.The viral genome has seven genes but encodes nine known viral proteins.The nucleocapsid protein (NP), phosphoprotein (P) and large RNApolymerase (L) protein are important for transcription and replicationof the viral RNA genome (Elango et al., 1988, J Gen Virol; 69(Pt11):2893-2900; Okazaki et al., 1992, Virology; 188:926-930; and Rima etal., 1980, J Gen Virol; 46(2):501-505). The V/P gene encodes threeproteins, I, V and P (Paterson and Lamb, 1990, J Virol; 64:4137-4145).Mutations in the P gene have been associated with increased virulence ofmumps virus (Saito et al., 1996, Microbiol Immunol; 40(4):271-275). TheV protein plays important roles in inhibiting interferon signaling ininfected cells (Kubota et al., 2002, J Virol; 76(24):12676-12682;Takeuchi et al., 1990, Virology; 178:247-253; Ulane et al., 2003, JVirol; 77(11):6385-6393; and Yokosawa et al., 2002, J Virol;76(24):12683-12690). The fusion (F) protein, a glycoprotein, mediatesboth cell-to-cell and virus-to-cell fusion in a pH-independent mannerthat is essential for virus entry into cells (Waxham et al., 1987,Virology; 159:381-388). The hemagglutinin-neuraminidase (HN), anotherviral glycoprotein, is also involved in virus entry (Tanabayashi et al.,1992, Virology; 187:801-804) and mutations in the HN gene have beenimplicated in mumps virus virulence (Cusi et al., 1998, J ClinMicrobiol; 36(12):3743-3744). The matrix (M) protein plays an importantrole in virus assembly (Matsumoto, 1982, Microbiol Immunol;26(4):285-320). The small hydrophobic (SH) protein is a 57-residue type1, hydrophobic integral membrane protein (Elango et al., 1988, J GenVirol; 69(Pt 11):2893-2900).

The present invention includes an isolated polynucleotide sequencerepresenting a mumps viral genome as described herein, and fragments andderivatives thereof. Such mumps viral genomes include, but are notlimited to, the wild type MuV-IA genome or a mumps viral genome lackingexpression of the viral proteins SH (rMuVΔSH) and/or V (rMuVΔV), andderivatives and fragments thereof. MuV, as a member of the familyParamyxoviridae, has a negative stranded, non-segmented RNA genome.Thus, in preferred embodiments, an isolated polynucleotide sequenceencoding the MuV-IA genome is a complementary DNA (cDNA). One such acDNA sequence is represented by SEQ ID NO:1. The genomic sequence of theMuV-IA virus, as well as the amino acid sequence of each encoded proteinmay be found on the National Center for Biotechnology Information (NCPI)website (available on the world wide web at ncbi.nlm.hih.gov) underGenBank Accession No. JN012242; Version JN012242.1 (GI:338784246), whichis herein incorporated by reference in its entirety. In someembodiments, an isolated polynucleotide representing the MuV-IA genomeis an RNA molecule. An isolated polynucleotide representing the MuV-IAgenome may be genome or antigenome RNA or cDNA. An isolatedpolynucleotide representing the MuV-IA genome may be a positive-senseversion of the MuV genome corresponding to the replicative intermediateRNA, also referred to as an antigenome.

Also included in the present invention are derivatives of an isolatedpolynucleotide described herein. In some embodiments, a derivativethereof may have at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% sequence identity to a polynucleotide sequence describedherein. For example, a derivative thereof may have at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NO:1, or a fragment thereof. In some embodiments, a derivativethereof may encode an amino acid sequence with at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to anamino acid sequence described herein, or encoded by a mumps viral genomedescribed herein. For example, a derivative thereof may encode apolypeptide sequence having at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity a polypeptide sequenceencoded by SEQ ID NO:1. Two polynucleotide sequences may be comparedusing the Blastn program of the BLAST 2 search algorithm, as describedby Tatusova and Madden, 1999, FEMS Microbiol Lett; 174:247-250), andavailable on the world wide web at ncbi.nlm.nih.gov/gorf/bl2.html.Preferably, the default values for all BLAST 2 search parameters areused, including reward for match=1, penalty for mismatch=−2, open gappenalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10,wordsize=11, and filter on.

In some embodiments, a derivative thereof hybridizes under “stringentconditions,” also referred to herein as “high stringency conditions,” toa polynucleotide sequence described herein. For example, a derivativethereof may hybridizes under stringent conditions to SEQ ID NO:1. Such aderivative thereof may further exhibit one or more of the variousfunctional traits described herein. Stringency of hybridizationreactions is readily determinable by one of ordinary skill in the art,and generally is an empirical calculation dependent upon probe length,washing temperature, and salt concentration. In general, longer probesrequire higher temperatures for proper annealing, while shorter probesneed lower temperatures. Hybridization generally depends on the abilityof denatured DNA to reanneal when complementary strands are present inan environment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature which can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995). “Stringent conditions” or “high stringencyconditions,” as defined herein, may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent,such as formamide, for example, 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

In some aspects, a derivative thereof includes the deletion and/oraddition of nucleotide sequences so that the derivative nucleotidesequences complies with “the rule of six.” See, for example, Kolakofskyet al., 1998, J Virol; 72:891-899.

Also included in the present invention are fragments of isolatedpolynucleotides, and derivatives thereof. Such fragments may includeonly a portion of the MuV genome, for example, encoding only one, two,three, four, five, six, seven, or eight of the nine mumps viralproteins. In some aspects, a fragment may serve as a primer or probe.

A fragment thereof may include a fragment of a mumps virus genomedetermined by any of the primer pairs described in Table 1 or Table 2.For example the fragment determined by any one of PX1F, PX3F, PX5F,PX7F, PX9F, PX11F, PX13F, PX15F, PX17F, PX19F, PX21F, PX23F, PX25F,PX27F, PX29F, PX31F, or PX33F paired with any one of PX2R, PX4R, PX6R,PX8R, PX10R, PX12R, PX14R, PX16R, PX18R, PX20R, PX22R, PX24R, PX26R,PX28R, PX30R, PX32R, or PX34R used as a primer pair in a PCR reactionwith a polynucleotide sequence described herein as a template. Forexample, a fragment of the present invention may represent the PCRproduct obtained when any one of PX1F, PX3F, PX5F, PX7F, PX9F, PX11F,PX13F, PX15F, PX17F, PX19F, PX21F, PX23F, PX25F, PX27F, PX29F, PX31F, orPX33F is used as a forward primer, and any one of PX2R, PX4R, PX6R,PX8R, PX10R, PX12R, PX14R, PX16R, PX18R, PX20R, PX22R, PX24R, PX26R,PX28R, PX30R, PX32R, or PX34R is used as a reverse primer on SEQ IDNO:1, or another mumps virus genome, including, but not limited to, anyof those described herein.

An isolated polynucleotide, derivative, or fragment thereof may includeadditional sequences not of mumps origin. Such heterologous sequencesmay, for example, encode additional antigenic determinants or otheradditional components, such as promoter, transcription initiation,and/or and termination sequences.

Included with the present invention are vectors and other constructsthat incorporate an isolated polynucleotide sequence encoding a mumpsvirus genome, such as MuV-IA, or a derivative, or fragment thereof. Sucha vector may be an expression vector. One such vector construct is aplasmid that includes the polynucleotide sequence encoding the completegenome of MuV, such as the MuV-IA. Such a plasmid is referred to hereinas a “pMuV.” The present invention includes a pMuV including any ofmumps genomes described herein. In some embodiments, the genome sequencemay be a cDNA sequence.

The present invention includes a reverse genetics system including amumps virus described herein, such as the MuV-IA genomic sequence, or amutant, or derivative thereof. Reverse genetics systems, as described inmore detail in the examples included herewith, can be used to generatein vitro infectious virus particles. See also, He et al., 1997,Virology; 237(2):249-60 and Tompkins et al., 2007, Virology;362(1):139-50. Such infectious viral particles are referred to herein asrecombinant MuV, also referred to herein as rMuV. A rMuV is produced byrecombinant means and is, thus, not naturally occurring. A rMuV mayfunction as an infectious viral particle. Included in the presentinvention are rMuV that express any of the mumps viral genomes describedherein. For example, a mumps viral genome unable to express a smallhydrophobic (SH) protein product and/or unable to express a V proteinproduct, including, but not limited to, the rMuVΔSH, rMuVΔV, orrMuVΔSHΔV constructs described herein.

A mumps viral genome as described herein, may belong to the G serotypeor the A serotype. A mumps viral genome may, for example, be the mumpsvirus strain MuV-IA, Glouc1/UK96(AF280799), UK01-22, 87-1004 (AF314560),SIPAR 02 (AF314558), Biken (AF314561), 87-1005 (AF314562), MuV (2001)(AF314559), Urabe 1004-10/2 (FJ375177), Urabe Gw7 (FJ375178), Hoshino(AB470486), Miyahara (1992) (NC_(—)002200), MuV Miyahara (1992) (2)(AB040874), Y213(AB576764), Dg1062/Korea/98 (32172464), L3/Russia/Vector(AY508995), L-Zagreb master seed (AY685921), L-Zagreb vaccine strain(AY685920), 9218/Zg98 (299766355), Novosbrisk genotype C (50404164),PetroNov genotype H (AY681495), 88-1961 (AF467767), Du/CRO05 (EU370207),SP-A (FJ556896), SP (EU884413), SP (2006) (DQ649478), JL2 (AF3452901),Jeryl Lynn sub strain (FN31985), Enders (GU9800521), Jeryl Lynn majorcomponent (AF338106), MuV (2000) (AF201473), JL1 (FJ211586), RIT4385(FJ211585), or RIT4385(2) (FJ211584). In some preferred embodiments, themumps viral genome is MuV-IA.

A mumps viral genome unable to express a small hydrophobic (SH) proteinproduct may include a deletion of the open reading frame (ORF) encodingthe SH protein or a mutation converting a start codon into a stop codon.For example, the deletion of the open reading frame (ORF) encoding theSH protein may include a deletion of about 156 nucleotides of the ORFencoding the SH protein.

A mumps viral genome unable to express a V protein product may includeone or more mutations to the V/I/P gene abrogating expression of the Vprotein. In some aspects, one or more mutations to the V/I/P geneabrogating expression of the V protein may include the nucleotidesequence GAGGAGGG at the editing site in the P/V gene.

A genome of a mumps virus of the present invention may include one ormore further mutations and/or deletions. In some aspects, a furthermutation or deletion may include a mutation or deletion effectingphosphorylation of the P protein. In some aspects, a further mutation ordeletion effecting phosphorylation of the P protein may include amutation or deletion at T147 and/or S307 of the P protein. Also includedin the present invention is a mumps virus genome, as described herein,further including sequences that allow for the expression of an Iprotein product. In some aspects, a further mutation or deletion mayinclude a mutation or deletion of the L gene. IN some aspects, a furtherdeletions and/or mutations may be selected from any of those know to oneof skill in the art.

The present invention also includes a mumps virus genome as describedherein, wherein the mumps genome further encodes a heterologouspolypeptide. Such a heterologous polypeptide may be for example, anantigenic polypeptide of non-mumps origin, or a detectable marker, suchas, for example GFP or luciferase.

Also included in the present invention are compositions including one ormore of the isolated polynucleotide sequences, pMuV, rMuV, vectorconstructs, infections viral particles, and/or viral constructs, asdescribed herein. Such a composition may include a pharmaceuticallyacceptable carrier. As used, a pharmaceutically acceptable carrierrefers to one or more compatible solid or liquid fillers, diluents orencapsulating substances which are suitable for administration to ahuman or other vertebrate animal. Carriers include, for example,stabilizers, preservatives and buffers. Suitable stabilizers include,for example, SPGA, carbohydrates (such as sorbitol, mannitol, starch,sucrose, dextran, glutamate or glucose), proteins (such as dried milkserum, albumin or casein) or degradation products thereof. Suitablebuffers include, for example, alkali metal phosphates. Suitablepreservatives include, for example, thimerosal, merthiolate andgentamicin. Diluents, include, but are not limited to, water, aqueousbuffer (such as buffered saline), alcohols, and polyols (such asglycerol). Such compositions and/or carriers may be pyrogen free.

Compositions of the invention may include an adjuvant, including, butnot limited to aluminum hydroxide; aluminum phosphate; QS-21 Stimulon;3-O-deacylated monophosphoryl lipid A; IL-12;N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine(CGP 19835A, referred to a MTP-PE); cholera toxin; and non-toxicderivatives of cholera toxin, including its B subunit; procholeragenoid,and fungal polysaccharides.

Compositions of the present invention may include additional activeimmunogens, including other immunologically active antigens againstother pathogenic species. The other immunologically active antigens maybe replicating agents or non-replicating agents. Replicating agentsinclude, for example, attenuated forms of measles virus, rubella virus,variscella zoster virus (VZV), Parainfluenza virus (PIV), andRespiratory Syncytial virus (RSV). Such an additional agent may be oneor more of those currently used in the combination measles-mumps-rubella(MMR) and measles-mumps-rubella-varicella (MMRV) vaccines. Theformulation of such compositions is well known in the art.

The present invention also includes methods of making and using theviral vectors and compositions described herein. The compositions of thepresent disclosure may be formulated in pharmaceutical preparations in avariety of forms adapted to the chosen route of administration. One ofskill will understand that the composition will vary depending on modeof administration and dosage unit. The agents of this invention can beformulated for administration in a variety of ways, including, but notlimited to, intravenous, topical, oral, intranasal, subcutaneous,intraperitoneal, intramuscular, and intratumor deliver. In some aspects,a composition is formulated for needle-less administration to themucosa, for example for intranasal administration to the upperrespiratory tract. It is expected that mucosal administration of thepharmaceutical composition to a mammalian subject will stimulate animmune response in mucosal tissues, including mucosal tissues that areremote from the site of administration, in addition to producing asystemic immune response in the subject.

The present invention also includes methods of inducing an immuneresponse in a subject by administering an isolated polynucleotidesequences, pMuV, rMuV, vector constructs, infections viral particles,viral constructs, or composition, as described herein to the subject.The immune response may or may not confer protective immunity. An immuneresponse may include, for example, a humoral response and/or a cellmediated response. Such an immune response may be a humoral immuneresponse, a cellular immune response, and/or a mucosal immune response.A humoral immune response may include an IgG, IgM, IgA, IgD, and/or IgEresponse. The determination of a humoral, cellular, or mucosal immuneresponse may be determined by any of a variety of methods, including,but not limited to, any of those described herein. The induction of animmune response may include the priming and/or the stimulation of theimmune system to a future challenge with an infectious agent, providingimmunity to future infections. The induction of such an immune responsemay serve as a protective response, generally resulting in a reductionof the symptoms. The immune response may enhance an innate and/oradaptive immune response. Immunogenicity may be assayed in any of avariety of animal models, including, but not limited to, mouse, ferret,and/or non-human primates model systems.

The isolated polynucleotide sequences, pMuV, rMuV, vector constructs,infections viral particles, viral constructs, or composition of thepresent invention may demonstrate reduced neurotoxicity whenadministered to a subject, for example, in comparison to mumps vaccinesin current use, such as, for example, the JL vaccine. Neurotoxicity maybe assayed by any of a variety of methods, including, but not limitedto, those in conventional use and any of those described herein,including a neurotoxicity test involving intracerebral inoculation intoneonatal rats (Rubin et al., 2000, J Virol; 74:5382-5384).

The present invention also includes methods of vaccinating a subject byadministering an isolated polynucleotide sequences, pMuV, rMuV, vectorconstructs, infections viral particles, viral constructs, orcomposition, as described herein to the subject. Such vaccination mayresult in a reduction or mitigation of the symptoms of future infectionand may prevent a future infection. Preferably, these compositions havetherapeutic and prophylactic applications as immunogenic compositions inpreventing and/or ameliorating mumps infection. In such applications, animmunologically effective amount of at least one attenuated recombinantmumps virus of this invention is employed in such amount to cause asubstantial reduction in the course of the normal mumps infection.Again, immunogenicity may be assayed in any of a variety of animalmodels, including, but not limited to, mouse, ferret, and/or non-humanprimates model systems. The isolated polynucleotide sequences, pMuV,rMuV, vector constructs, infections viral particles, viral constructs,or composition of the present invention may demonstrate reducedneurotoxicity when administered to a subject, for example, in comparisonto mumps vaccines in current use, such as, for example, the JL vaccine.Neurotoxicity may be assayed by any of a variety of methods, including,but not limited to, those in conventional use and any of those describedherein, including a neurotoxicity test involving intracerebralinoculation into neonatal rats (Rubin et al., 2000, J Virol;74:5382-5384).

With the methods of the present invention, any of a variety of modes ofadministration may be used. For example, administration may beintravenous, topical, oral, intranasal, subcutaneous, intraperitoneal,intramuscular, or intratumor. In some aspects, administration is theneedleless administration to a mucosal membrane, for example, by theintranasal administration to the upper respiratory tract by spray,droplet or aerosol

An agent of the present disclosure may be administered at once, or maybe divided into a number of multiple doses to be administered atintervals of time. For example, agents of the invention may beadministered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or moretimes, or may be administered by continuous infusion. It is understoodthat the precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by extrapolation from in vivo or in vitro testdata. It is to be noted that concentrations and dosage values may alsovary with the severity of the condition to be alleviated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that any concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed compositions and methods.

By a “therapeutically effective amount” is meant a sufficient amount ofthe compound to treat the subject at a reasonable benefit/risk ratioapplicable to obtain a desired therapeutic response. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including, for example, the disorderbeing treated and the severity of the disorder, activity of the specificcompound employed, the specific composition employed, the age, bodyweight, general health, sex and diet of the patient, the time ofadministration, route of administration, and rate of excretion of thespecific compound employed, the duration of the treatment, drugs used incombination or coincidentally with the specific compound employed, andlike factors well known in the medical arts.

In some therapeutic embodiments, an “effective amount” of an agent is anamount that results in a reduction of at least one pathologicalparameter. Thus, for example, in some aspects of the present disclosure,an effective amount is an amount that is effective to achieve areduction of at least about 10%, at least about 15%, at least about 20%,or at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95%, compared to the expected reduction in theparameter in an individual not treated with the agent.

As used herein, the term “subject” includes, but is not limited to,humans and non-human vertebrates. In preferred embodiments, a subject isa mammal, particularly a human. A subject may be an individual. Asubject may be an “individual,” “patient,” or “host.” Non-humanvertebrates include livestock animals, companion animals, and laboratoryanimals. Non-human subjects also include non-human primates as well asrodents, such as, but not limited to, a rat or a mouse. Non-humansubjects also include, without limitation, chickens, horses, cows, pigs,goats, dogs, cats, guinea pigs, hamsters, ferrets, mink, and rabbits.

As used herein “in vitro” is in cell culture and “in vivo” is within thebody of a subject. As used herein, “isolated” refers to material thathas been either removed from its natural environment (e.g., the naturalenvironment if it is naturally occurring), produced using recombinanttechniques, or chemically or enzymatically synthesized, and thus isaltered “by the hand of man” from its natural state.

As used herein, an “isolated” substance is one that has been removedfrom its natural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. For instance, a polypeptide, apolynucleotide, or a cell can be isolated. Preferably, a substance ispurified, i.e., is at least 60% free, preferably at least 75% free, andmost preferably at least 90% free from other components with which theyare naturally associated.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded RNA and DNA. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidemay be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. A polynucleotide may include nucleotidesequences having different functions, including, for instance, codingregions, and non-coding regions such as regulatory regions.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “the,” and “at least one” are usedinterchangeably and mean one or more than one.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

The description exemplifies illustrative embodiments. In several placesthroughout the application, guidance is provided through lists ofexamples, which examples can be used in various combinations. In eachinstance, the recited list serves only as a representative group andshould not be interpreted as an exclusive list.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Rescue of Wild-Type Mumps Virus from a StrainAssociated with Recent Outbreaks Defines Role of the SH ORF in thePathogenesis of Mumps Virus

With this example, the complete genome of a representative strain fromthe epidemic (MuV-IA) was sequenced. MuV-IA is a member of genotype G,the same genotype of MuV that was associated with the outbreak in the UKin 2004-2005. A reverse genetics system was constructed for MuV-IA(rMuV-IA) and used to rescue a virus lacking the open reading frame(ORF) of the SH gene (rMuVΔSH). rMuVΔSH infection in L929 cells inducedincreased NF-κB activation, TNF-α production and apoptosis compared torMuV-IA. rMuVΔSH was attenuated in an animal model. These resultsindicated that the SH ORF of MuV plays a significant role in interferingwith TNF-α signaling and viral pathogenesis during virus infection.

Results

Sequence of the complete genome of MuV-IA. To better understand thegenetic characteristics of viruses associated with recent outbreaks inthe U.S., the complete genomic sequence of a representative isolate fromthe Iowa outbreak was determined. It is available as GENBANK AccessionNo. JN012242. A set of primers was designed based on the consensussequence derived from comparison of the genomic sequences of Jeryl Lynn,Urabe, 88.1961 and PetroNov. These primers are shown in Table 1. ViralRNA of MuV-IA was reverse-transcribed into cDNA using random hexamers,PCR reactions were then carried out using the set of primers and theproducts were sequenced using the corresponding primers. A second set ofprimers based on the sequencing results were then used to perform RT-PCRand the products overlapping with those of first round of sequencingfragments were sequenced using the primers. This second set of primersis shown in Table 2. Leader and trailer sequences were determined byperforming 5′/3′ RACE.

TABLE 1 Mumps virus specific primers Approximate SEQ genomicprimer sequence ID Primer location (5′ → 3′) NO: PX1F 100-300ATGTCGTCCGTGCTCAAAG 48 PX2R 1100-1300 CGGTCTCAACCCCAATCTG 49 PX3FGGGGGCTACCCATTGATATT 50 PX4R 2100-2300 GAAAAGGGGCTCAGGAATCT 51 PX5FTTCAGTACCCCACTGCATCA 52 PX6R 3100-3300 GGCTGGATTGGACTTGTGTT 53 PX7FCGAGGATGCCCTGAATGATA 54 PX8R 4100-4300 GCATAGTCTGAGCCCTGGAG 55 PX9FCACATTCCGACAACTGCAAA 56 PX10R 5100-5300 TGAACCACTGCAGGTGTCAT 57 PX11FGCTTGCAACCTCCCTAGGAT 58 PX12R 6100-6300 TGGCACTGTCCGATATTGTG 59 PX13FGTGTCGATGATCTCATCAGGTACT 60 PX14R 7100-7400 ACCTCAAAGCACGCTGATCT 61PX15F GGGAATTGGGCTACTTTGGT 62 PX16R 7900-8300 GTGCATGAACCTGGGATTCT 63PX17F GATACCGGTGATGCTAGTGTG 64 PX18R 9100-9300 GAAAGAAAGCCAGGGTCTTCA 65PX19F GCTCTACTCATGGGGGACAA 66 PX20R 10100-10300 ATCAAGGTCAAGTTGGGTAGGA67 PX21F CCAAGTCATCATCCCCTTTG 68 PX22R 11100-11500 TTGCTGACAATGGTCTCACC69 PX23F CATGCCCAATATACATTGATGG 70 PX24R 12100-12300TGAAGGGTACAGGAAGCAAAG 71 PX25F CTGGCCTTGCTTTAATTGAGA 72 PX26R13100-13300 AGAGATGCTGATTCGGATGAA 73 PX27F GAACCAAAATTAACTGCCTACCC 74PX28R 14100-14300 CCGCCTGAAGGATAATGTTG 75 PX29F CCCTGAATGAACAGGGGTTT 76PX30R 15100-15300 CTTTTGCTGGCCTTTTGCT 77 PX31F CTGCTAACAAAGCCCGAAAG 78PX32R 16100-16300 AAGTTGCAGGACCACTTCTG 79 PX33F TGACTCCCCGTCGTGTAGAT 80PX34R 17100-17300 AGACGTCAGGTGGCACTTTT 81

TABLE 2 MuV-IA primers SEQ ID Primer Location Sequence (5′ → 3′) NO: 5FIowa-L- TGAATCATAGTGAATGCAGCAGG 26 upstream-1 6F Iowa-L-GCCCTATTGGCGTGTCTCA 27 upstream-2 7R Iowa-L- TGGTGACGTATCGTGCCAGA 28downstream 10F Iowa-NP-F AACAGTAAGCCCGGAAGTG 29 11R Iowa-NP-RCCAATGAGTACTGGTGCAAC 30 12F Iowa-P-F GCGACTGGGATGAGTAAA 31 13R Iowa-P-RTGGATTGGACTTGTGTTCG 32 14F Iowa-M-F GCGAGACATCATACGAAG 33 15R Iowa-M-RAAGCTTGACCACTATGTAGG 34 16F Iowa-F-F CCTCAATGAGCAACCTATG 35 17R Iowa-F-RTTAGTACCTGATGAGATCATCG 36 18F Iowa-SH- GAATTCATGCCGGCGATCCAAC 37 F-EcoRI19R Iowa-SH- GCTAGCTTAGAGTGAGTGATCGAAAC 38 R-NheI 20F Iowa-HN-FATGGAGCCCTCGAAATTCT 39 21R Iowa-HN-R AACGATGGGTGAGTTTAAATG 40 22FIowa-NP-F2 GGCTTGGGTGATGGTCTGTA 41 23R Iowa-NP-R2 CATTTTGGAATCCTGCACCT42 24F Iowa-HN-F2 TGCAAGGACCATACTTCGTC 43 25R Iowa-HN-R2GAGTTCATACGGCCACCAG 44 26F Iowa-P-F2 CTCAACGCCGGTAACAGAAT 45 27FIowa-F-F2 ATGAAGGTTCCTTTAGTTACTTGC 46 28F Iowa-P-F3 AGCCAACTGCTCAAATCCAC47

There is only one conserved change in the putative transmembrane domainof the SH protein when the SH protein sequence of MuV-IA was compared toother strains of mumps virus in genotype G (FIG. 1A), confirming thatMuV-IA belongs to genotype G (Rota et al., 2009, J Med Virol;81(10):1819-1825). To further study the genomic divergence of MuV-IA, aphylogenetic tree was generated using the genomic sequence of MuV-IA and32 full length genomic sequences from Genbank (FIG. 1B). Phylogeneticanalysis indicated that MuV-IA is most closely related to the sequenceof MuV Du/CRO05, a genotype G virus, which was isolated in Croatia in2005 (Santak et al., 2006, J Med Virol; 78(5):638-643). A comparison ofthe predicted amino acid sequences between the protein coding regions ofMuV-IA and Jeryl Lynn vaccine (major component) showed that while NP, Mand L protein sequences are highly conserved with an identity of over98%, there was more divergence among V, P, F, SH and HN proteins (FIG.1C). The predicted SH protein sequences had only 85% identity.

Generation of an infectious cDNA clone for MuV-IA. To study thepathogenesis of MuV-IA, a reverse genetics system was derived. BecauseRNA viruses exist as a quasi-species, the consensus sequence of thegenome was used as the base for the recombinant MuV. A plasmidcontaining a mini-genome with luciferase (Luc) reporter gene for mumpsvirus (pT7-MuV-Mini-Luc) similar to the PIV5 mini-genome expressingplasmid was constructed using rMuV-IA trailer and leader sequences (Linet al., 2005, Virology; 338(2):270-280). In addition, plasmids encodingNP, P and L in the pCAGGS vector have been obtained and confirmed bysequencing. To test the functionality of the plasmids, the plasmids weretransfected into BSRT7 cells. At 2 dpi, the cells were harvested andLuciferase (Luc) assays were performed. Luc activity was detected in thecell transfected with all plasmids, not ones missing P or L, indicatingthat the plasmids expressed functional P and L proteins. RT-PCR wasconducted to amplify DNA fragments representing the complete genome andinserted into individual plasmid vectors before being assembled into afull-length genome. The plasmid with the full length genome of MuV-IAexpressed under the control of a T7 (pMuV-IA) promoter (pMuV-IA) wassimilar to the plasmid used to generate infectious PIV5 (He et al.,1997, Virology; 237:249-260). pMuV-IA had changes in two nucleotideswithin the L ORF compared with consensus sequence of MuV-IA at positionsof 11863 (T to C) and 12028 (C to T). However, neither of thesenucleotide changes resulted in changes in the predicted L proteinsequence. A recombinant MuV (rMuV-IA) was rescued using the plasmidcontaining the full-length genome of MuVIA. BSRT-7 cells wereco-transfected with pMuV-IA and plasmids expressing viral RNA polymerasecomponents. Individual plaques were selected and amplified in Verocells. The entire genome of the rescued virus was sequenced and found tomatch the input cDNA genome sequence.

To compare time course of the growth of rMuV and MuV-TA, a multi-cyclegrowth assay was performed (FIG. 2A). Both viruses grew to similar peaktiters in Vero cells. Viral titers in the supernatant of the infectedcells increased exponentially during the first two days after infection,and reached a titer of 10⁷ pfu/ml at 48 hpi. The growth of both virusesin HeLa cells (a human cell line), MDBK cells (a bovine cell line), andL929 cells (a murine cell line) was also compared, and no obviousdifferences between these two viruses were observed. The viral proteinexpression levels in cells were also examined using Western blot (FIG.2B) and the protein levels were similar at different time points afterinfection, indicating that the replication of rMuV resembles MuV-IA intissue culture cells.

In addition, infectious recombinant viruses expressing either EGFP orRenila Luciferase (RL) protein as an extra gene were rescued. pMuVEGFPwas constructed by inserting an EGFP gene, flanked by gene start (GS) ofSH and gene end (GE) of NP, between F gene and SH gene in pMuV-IA,pMuV-RL was constructed through substitution of coding sequence of EGEPwith that of renilla luciferase (RL) in pMuV-EGFP. Expression of EGFP orRL in the infected Vero cells was detected (FIGS. 2C and 2D).

Rescue of a recombinant mumps virus lacking the SH ORF. To study thefunction of the SH protein of MuV, 156 nucleotides in the SH gene openreading frame (ORF) of the SH gene were deleted from pMuV-IA. Thetruncated SH ORF contained a short ORF encoding five amino acid residuesflanked by the original SH ORF start and gene end (pMuV-IAΔSH, FIG. 3A).An infectious MuV lacking the SH ORF was rescued (rMuVΔSH) (FIGS. 3B and3C) and the genome was sequenced, which matched the input cDNA sequence.The rMuVΔSH genome was of 15,228 nt in size, complying with “the rule ofsix” (Kolakofsky et al., 1998, J Virol; 72:891-899). To confirm thatwtMuV and rMuV did express a SH protein and that rMuVΔSH did not, celllysates of infected Vero cells were examined by immunoblotting withanti-SH as well as anti-NP and anti-P (FIG. 3D). SH was detected inMuV-IA and rMuV-infected cells, but not in rMuVΔSH-infected cells,confirming the lack of the SH protein expression in rMuVΔSH-infectedcells.

Analysis of rMuV and rMuVΔSH. To investigate the growth rate of rMuVΔSH,a multiple-cycle growth curve and protein expression levels wereexamined in Vero cells, and the titers of the viruses released fromrMuVΔSH-infected Vero cells remained similar to rMuV-infected Vero cellsat all time points (FIG. 4A). When the infected cells were lysed andviral protein expression levels were compared using Western blot, theprotein levels of NP and P in rMuVΔSH and rMuV-infected cells weresimilar (FIG. 4B), indicating that the SH ORF was not essential forviral gene expression, or virus release in Vero cells, consistent withthe previous findings. The HN gene is downstream of the SH gene. Toexamine whether there is any significant impact of the deletion of theSH ORF on the expression level of HN, expression levels of HN and NP ofinfected cells were examined using flow cytometry. As shown in FIG. 4C,relative expression level of HN in rMuV-infected cells and inrMuVΔSH-infected cells were similar, suggesting that the deletion of theSH ORF sequence did not affect expression of the HN protein.Furthermore, mRNA expression levels of HN were examined using real-timeRT-PCR. No significant difference was observed between rMuV and rMuVΔSH(FIG. 4D). Interestingly, rMuVSH formed larger plaques in Vero cellscompared to rMuV (FIG. 4A).

rMuVΔSH induced cytopathic effect in L929 cells. We compared infectionof Vero, MDBK and HeLa cells with rMuVΔSH and rMuV. At one day postinfection, there were no observable differences in rMuVΔSH- orrMuV-infected Vero and MDBK cells. Previous studies in our lab showedthat the SH ORFs of PIV5 and RSV played a role in blocking TNF-αsignaling. To test the hypothesis that mumps virus SH ORF has a role inregulating the TNF-α signaling pathway, the phenotype of rMuVΔSH in L929cells, which undergo apoptosis after TNF-α treatment, was investigated.rMuVΔSH infection led to significantly more cell death than infectionswith rMuV or wtMuV. The phenotype was evident at 2-day post infection(FIG. 5B). To investigate whether the cytopathic effects (CPE) observedin rMuVΔSH infected L929 cells was caused by apoptosis, TUNEL assay wasperformed. At 1 dpi, infection with rMuVΔSH resulted in a higherpercentage of infected cells with apoptosis than rMuV (FIG. 5C),indicating that the lack of SH led to increased apoptosis in infectedcells.

TNF-α played a critical role in rMuVΔSH-induced apoptosis. To testwhether apoptosis in rMuVΔSH infected L929 cells resulted from anelevated TNF-α, the activation of NF-κB in rMuVΔSH-infected L929 cellswas examined by examining nuclear translocation of p65, a key subunit ofNF-κB. NF-κB factors are localized in the cytoplasm. On activation, forexample by TNF-α stimulation, p65 is translocated into the nucleus (Baudand Karin, 2001, Trends Cell Biol; 11(9):372-377). A higher level of p65nuclear localization was observed in rMuVΔSH-infected L929 cells (FIG.6A), indicating activation of NF-κB. To investigate whether theproduction of TNF-α was increased in rMuVΔSH-infected cells,supernatants of infected were collected and levels of TNF-α weremeasured using ELISA. TNF-α production level was up regulated inrMuVΔSH-infected cells (FIG. 6B). To determine whether the increasedTNF-α played a role in increased apoptosis in rMuVΔSH infected cells,the infected cells were treated with neutralizing antibody againstTNF-α. Anti-TNF-α reduced CPE in rMuVΔSH infected cells, while thecontrol antibody had no effect (FIG. 6C), indicating that TNF-α played acritical role in rMuVΔSH induced cell death. This was confirmed withTUNEL assay (FIG. 6D). At 1 dpi, with control antibody treatment,rMuVΔSH induced almost 4-fold higher apoptotic rate than rMuV. Treatmentof anti-TNF-α antibody effectively blocked cell death in infected cells(FIGS. 6C, D).

SH of MuV-IA blocked TNF-α signaling in vitro. To investigate whetherMuV-IA SH expressed alone can block TNF-α signaling, a plasmid encodingSH of MuV-IA was co-transfected with a NF-κB promoter-luciferasereporter system into L929 cells. At one day post transfection, cellswere treated with TNF-α. TNF-α signaling was blocked by SH of MuV-IA aswell as SH of PIV5, but not by NP of MuV-IA (FIG. 7A) or the sequence ofthe SH ORF (FIG. 7B), indicating that the SH protein can blockTNF-α-mediated signaling.

rMuVΔSH was attenuated in vivo. MuV is a human virus and there is noideal animal model in which to study viral pathogenesis. Intracerebralinjection of MuV into newborn rats has been used to compare the relativepathogenecities of different strains of MuV (Rubin et al., 2005). Tocompare the neurotoxicity of the viruses, rMuV or rMuVΔSH was injectedintracerebrally into brains of newborn rats. Relative neurotoxicityscore was calculated based on relative severity of hydrocephalus. Asshown in FIG. 8, rMuVΔSH had a lower neurotoxicity score than rMuV,indicating that deletion of the SH ORF resulted in attenuation in vivo.

Discussion

Immunization against MuV is a part of a 2-dose MMR (mumps, measles andrubella) vaccine regimen that is administrated to children at 1 and 5years of age in the U.S. Even with a two-dose vaccination schedule,large outbreaks have occurred in vaccinated populations. This exampledescribes the rescue of a wild-type mumps virus that is representativeof the strain associated with recent outbreaks in the U.S. and Europe.This example identifies the potential role of the SH protein inregulating TNF-α, and demonstrates that the deletion of the SH ORFresulted in attenuation in vivo, indicating that SH plays a role inviral pathogenesis. The attenuation of rMuVΔSH in vivo suggests thatdeleting the SH ORF can be a possible strategy to develop attenuatedmumps strains. Recombinant MuVs expressing foreign genes such as GFP andRL have been obtained, and interestingly, the expression level of RL inrMuV-RL in Vero cells remained relatively high after 20 passages,indicating that MuV can possibly be used as a vector.

The SH protein of paramyxoviruses was first identified in PIV5-infectedcells (Hiebert et al., 1985, J Virol; 55:744-751). A similar gene waspredicted basing on sequence analysis of the Enders strain of MuV.However, due to a mutation in the intergenic sequence of the putative SHgene, the SH protein of the Enders strain MuV is not expressed ininfected cells (Takeuchi et al., 1991 , Virology; 181:364-366). Thus,the SH protein of MuV has never been detected in MuV-infected cells.Wilson et al. replaced the SH ORF within the genome of PIV5 with the SHORF of MuV Enders strain and found that the MuV SH can functionallyreplace the SH ORF of PIV5 (Wilson et al., 2006, J Virol;80(4):1700-09). Thus, it is thought that the function of MuV SH is thesame as the function of the SH ORF of PIV5, a closely relatedparamyxovirus. In this example, the expression of SH was detected inMuV-infected cells for the first time, confirming the existence of theSH protein in MuV-infected cells. Furthermore, taking advantage of thenew reverse genetics system, a recombinant MuV lacking the SH ORF(rMuVΔSH) was obtained and analyzed.

One interesting observation was that rMuVΔSH produced larger plaques. Apossible explanation is that the deletion of the SH ORF resulted in avirus that promotes cell-to-cell fusion better than the wild type virus.Because there was no change of total number of ORFs or the overall orderof genes, we expect that the relative amounts of viral mRNAs and theexpression levels of viral proteins of rMuVΔSH should be similar tothose of wild type virus (FIGS. 4B, 4C, and 4D). Thus, it is unlikelythat the bigger plaque formation by rMuVΔSH was due to a higher level ofviral protein expression. Further, a fusion assay using cellstransfected with MuV HN and F was performed in the presence or absenceof MuV SH, and no difference in the extent of cell-to-cell fusion wasobserved, suggesting that the SH does not have a role in promotingcell-to-cell fusion. It is possible that the larger plaques farmed byrMuΔSH are due to a higher level of induction of cell death by rMuVΔSH.The viruses infected cells at the same rate; however, the cells infectedby rMuVΔSH induced more cell death than rMuV resulting in more rapidcell death at the edge of a plaque.

It is possible that the mRNA of from some ORFs may have biologicfunctions. For example, the mRNA of the L ORF of PIV5 is capable ofactivating IFN-β expression (Luthra et al., 2011, Proc Natl Acad SciUSA; 108(5):2118-2123). In this example, the ORF of SH was deleted, andthe function of the polypeptide encoded by the SH ORF cannot bedifferentiated from SH mRNA itself. While the SH polypeptide was neededto block TNF-α mediated signaling, not the sequence of the SH ORF, andwe favor a critical role of the SH polypeptide in mumps viruspathogenesis; however, it is possible that the small mRNA potentiallyexpressed from the deleted SH gene could have contributed to thephenotype of rMuVΔSH. The reduced neurotoxicity of rMuVΔSH in neonatalrat brain indicates that the SH ORF plays a critical role in viralpathogenesis. We propose that infection with rMuVΔSH induced a higherlevel of proinflammatory cytokine expression, resulting in a more rapidresolution of infection, thus limiting damage in the infected brain.

Material and Methods

Plasmids, viruses and cells. All molecular cloning was conductedaccording to standard procedures as previously described (He et al.,1997, Virology; 237:249-260). MuV-IA NP, P and L genes were cloned intothe pCAGGS expression vector (Niwa et al., 1991, Gene; 108:193-200).MuV-IA SH gene was cloned into the pCAGGS expression vector. MuV-IA SH(stop codon) was constructed by introducing three continues stop codonsequence into the SH ORF, six nucleotides downstream of the start codon.Construction of MuV-IA full-length cDNA in pUC19 was analogous to thePIV5 reverse genetics system (He et al., 1997, Virology; 237:249-260).To construct pMuVΔSH, the region of the SH ORF from the 4th amino acidto the 57th (156 nt) was substituted with a short six nucleotidesequence designed to facilitate subcloning and to maintain the length ofthe genome a multiple of six (known as the “rule of six”). pMuV-EGFP andpMuV-RL were constructed by inserting either an EGFP or a renillaluciferase gene between F and SH gene flanked by F gene start and SHgene end.

To rescue an infectious virus from cDNA, plasmid (5 μg) containing afull-length genome or a mutated MuV genome was co-transfected withplasmids pCAGGS-L (1 μg), pCAGGS-NP (1.5 μg) and pCAGGS-P (200 ng) intoBSRT-7 cells. Usually four to seven days post-transfection, syncytiaformation could be observed in transfected BSRT-7 cells. Supernatantswere plagued in Vero cells. Plaques could be visualized at 4 to 7 dpi.One or two plaques from each independent rescue were amplified in Verocells and their genomes were sequenced.

Vero, HeLa, MDBK and L929 cells were maintained in Dulbecco's modifiedEagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1%penicillin-streptomycin (P/S) (Mediatech Inc., Holu Hill, Fla.), BSRT-7cell were maintained in DMEM supplemented with 10% FBS, 1% P/S and 10%tryptose phosphate broth (TPB) plus G418 at 400 μg/ml. Cells werecultured at 37° C. with 5% CO₂ and passed the day before infection ortransfection at appropriate dilution factors to archived 80.90%confluence the next day. For virus infection, cells were infected withviruses in DMEM plus 1% bovine serum albumin (BSA) at MOI of 0.01, 3 or5 and incubated for 1 to 2 h at 37° C. with 5% CO₂. The culturing mediumwas then replaced with DMEM supplied with 2% FBS and 1% P/S. Fortransfection, cells were transfected with plasmids using PLUS™ andLipofectamine™ reagents from Invitrogen following the manufacturerprovided protocol.

MuV-IA (MuV/Iowa/US/2006) was isolated at the Iowa Hygenic Laboratoryfrom a buccal swab obtained from a mumps case during the early phase ofthe outbreak in 2006. Genotype analysis was performed at the CDC (Rotaet al., 2009, J Med Virol; 81(10):1819-1825) and the accession numberfor the SH sequence is DQ661745. All mumps viruses were grown in Verocells and were harvested 4 to 7 dpi. Virus titers were measured in Verocells by plaque assay followed by Giemsa staining as described before(He and Lamb, 1999, J Virol; 73:6228-6234; and He et al., 1997,Virology; 237:249-260).

Sequencing of viruses. Viral RNA was extracted from cell culturesupernatants using QIAampR viral RNA extraction mini kit from QIAGENfollowing manufacturer's protocol. Isolated total RNA was reversetranscribed into cDNA using Super ScriptR III reverse transcriptase fromInvitrogen with random hexamers. Synthesized cDNA was then served astemplates for PCR using mumps virus genome specific primers (Table 1)and Taq polymerase from Invitrogen. Fifteen sets of primers (shown inTable 2), each contained a forward and a reverse primer, were designedas to divide the genome into fifteen overlapped fragments. The primerswere used for the subsequent sequencing of the PCR products (Li et al.,2011, J Virol; 85(1):32-42). Leader and Trailer sequences were sequencedfollowing standard protocol of Rapid Amplification of cDNA Ends (RACE)(Li et al., 2011, J Virol; 85(1):32-42).

Generation of monoclonal and polyclonal antibodies against mumps NP, Pand SH. To generate monoclonal antibodies against MuV-IA, the virus wasgrown in Vero cells. The medium of infected Vero cells was collected,and clarified with low-speed centrifugation at 3 K rpm for 10 min. Theclarified media containing virus was overlaid onto a 10 ml 20% sucrosesolution and centrifuged at 40 K rpm for 1.5 h at 4° C. The pellet wasresuspended in 0.5 ml 10×PBS, mixed with 1.3 ml 80% sucrose solution andoverlaid by a decreasing sucrose gradient from bottom to top: 1.8 ml 50%sucrose solution and 0.6 ml 10% sucrose solution. The sucrose gradientwith virus at the bottom was centrifuged at 45 K rpm for 3 hours (h) at4° C. 1 ml fractions were collected, mixed with 10 ml 1×TEN buffer (100mM NaCl, 10 mM Tris-base, 1 mM EDTA) and spun down at 40 K rpm for 1.5 hat 4° C. The pellet containing virus was suspended in 50 μl of 1×TENbuffer plus 1% NP-40 and used for generation of mouse hybridoma cells.Mouse hybridoma cells generating monoclonal antibodies against MuV-IA NPand P were engineered by the core facility in the Pennsylvania StateUniversity. The hybridomas were culture in D-MEM supplied with sodiumpyruvate, with addition of 20% FBS and 0.1% Gentamicin at 37° C. with 5%CO₂.

To generate polyclonal antibodies against MuV-IA SH, two peptides(N-terminal MPAIQPPLYLTFLLC (SEQ ID NO:10) and C-terminalCYQRSFFHWSFDHSL (SEQ ID NO:11)) were purchased from GenScriptCorporation. Two peptides (QFIKQDETGDLIETC (SEQ ID NO:12) andCSRPDNPRGGHRREW (SEQ ID NO:13)) were used to generate polyclonalantibodies against MuV-IA V (GenScript Corporation) in rabbits.

Treatment of infected cells with anti-TNF-α. L929 cells in six wellplates were infected with rMuVΔSH or rMuV at a MOI of 5 and cultured inDMEM supplemented with 2% FBS and 1% P/S with neutralizing anti-TNF-αantibody or control antibody (BD Pharmingen) at 50 μg/ml for 1 or 2days. At 1 day or 2 dpi, cells were photographed with a microscope witha digital camera, and then collected for MuV-NP staining or TUNEL assay.

Flow cytometry and TUNEL assay. Flow cytometry was performed aspreviously described (Timani et al., 2008, J Virol; 82(18):9123-9133).L929 cell in 6 well plates were infected with rMuVΔSH or rMuV or mockinfected at MOI of 3 or 5. At 1 or 2 dpi, attached cells weretrypsinized and combined with floating cells in the culture media. Cellswere centrifuged and resuspended in 0.5% formaldehyde in phosphatebuffered saline (PBS) for one hour at 4° C. The fixed cells were thenwashed with PBS, permeabilized in 50% FCS-50% DMEM plus three volumes of70% ethanol overnight. Permeabilized cells were subjected for eitherTUNEL staining for apoptotic cells according to manufacturer's protocolor MuV-NP staining for infection rate. When cells were for NP staining,monoclonal MuV-NP antibody was diluted to 1:200 followed by PEanti-mouse secondary antibody staining at a dilution factor of 1:100.

Vero cells were mock infected or infected with rMuVΔSH or rMuV at a MOIof 0.5 or 0.01. At 24 or 48 hpi, attached cells were collected incombination with floating cells, fixed. For HN surface staining, cellswere directly stained with anti-HN at a dilution factor of 1:50; fortotal staining of HN and NP staining, fixed cells were permeabilizedwith 0.1% saponin in PBS and stained with anti-NP at a dilution factorof 1:200 or anti-HN at a dilution factor of 1:50.

Assays for detection of activation of NF-κB. L929 cells on glass coverslips in six well plates were infected with rMuVΔSH, or rMuV at MOI of0.01, or mock infected. At 2 dpi the cover slips were washed with PBS,and fixed in 0.5% formaldehyde. The fixed cells were permeabilized withPBS plus 0.1% saponine and then incubated with mouse anti-P65 (SantaCruz Biotechnology) in PBS with 0.1% saponine followed by secondary FITClabeled goat antimouse antibody (Jackson Laboratory). The cells werephotographed using a fluorescence microscope with a digital camera.

The NF-κB reporter assay system was performed as described previously(Wilson et al., 2006, J Virol; 80(4):1700-09). L929 cells were platedinto 24 well plates and transfected using PLUS™ and Lipofectamine™reagents with either empty vector, pCAGGS-MuV SH, pCAGGS-MuV SH (stop),pCAGGS-PIV5 SH or pCAGGS-MuV NP, plus a picB-TATA-Luc (a reporterplasmid containing a NF-κB promoter region followed by TATA box enhancerand a firefly luciferase gene) and a pCAGGS-RL (a transfection controlplasmid expressing renilla luciferase protein). On the second day posttransfection, half of the cells were treated with TNF-α (Alexis, SanDiego) at a concentration of 10 ng/ml in Optima (Invitrogen) for 4 h at37° C. with 5% CO₂; half of the cells were treated with Optima only.Cells were then lysed with 100 μl 1× passive lysis buffer (Promega,Madison, Wis.) and 10 μl of the lysate were subjected for dualluciferase assay using a dual luciferase assay kit (Promega, Madison,Wis.). The ratio of TNF-α stimulated cells over no TNF-α stimulation isused as “induction of luciferase activity.”

Immunoblotting. Vero cells in 6 well plates at about 90% confluence wereinfected with mock, MuV-IA, rMuV or rMuVΔSH at a MOI of 0.05. Cells werecollected and lysed at 0 h, 24 h, 48 h, or 72 h post-infection in 0.5 mlWCEB buffer (50 mM Tris.HCl PH 8.0, 120 mM NaCl, 0.5% NP-40, 0.00076%EGTA, 0.2 mM EDTA, 10% Glycerol) with a mixture of protease inhibitorsas described before (Luthra et al., 2008, J Virol; 82(21):10887-10895).Cell lysates were briefly centrifuged to remove cell debris. Celllysates were loaded into 10% or 17.5% polyacrylamide gel and subjectedfor SDS-PAGE. Protein were transferred to Immobilon-FL transfer membrane(Millipore), incubated with primary antibody (anti-MuV SH 1:250,anti-MuV V 1:500, anti-MuV NP 1:5000, anti-MuV P 1:2000) andcorresponding secondary antibodies conjugated to horseradish peroxidase,and detected by Amersham ECL™ western blotting detection kit (GEHealthcare).

Time course of rMuVΔSH, rMuV and MuV-IA infection in cell culture. Cellsin 6 cm plates were infected with MuV-IA, rMuV, or rMuVΔSH at MOI of0.01. 100 μl of supernatant were collected at 0 h, 24 h, 48 h, 72 hpostinfection and frozen down at −80° C. supplemented with 1% BSA. Virustiters were determined by plaque assay using Vero cells in 24 wellplates in triplicates. After one to two hours incubation with theviruses, growth media were changed into semisolid DMEM with 2% FBS, 1%P/S and 1% low melting point agarose. 4 to 7 dpi, 24 well plates of Verocells were stained with Giemsa stain and plaques were counted.

Enzyme-linked immunosorbent assay (ELISA) of TNF-α. L929 cells in 6 wellplates were infected with mock, rMuV or rMuVΔSH at MOI of 5. Culturingmedia were collected at 1 dpi, 2 dpi and 3 dpi. The amount of TNF-αsecreted into the culturing media was measured using a murine TNF-αdetection kit (Amersham Pharmacia) following the procedures describedbefore (Li et al., 2011, J Virol; 85(1):32-42).

Real time RT-PCR. Vero cells were mock infected or infected with rMuVΔSHor rMuV at a MOI of 0.005. Viral RNA was extracted from infected cellsat 4 dpi using QIAGEN RNeasymini kit and reverse transcribed into cDNAusing Oligo-dT as primers. MuV F and HN mRNA specific FAM tagged probeswere purchased from Applied Biosystems™. Real time PCR was assembledusing TaqMan® Gene Expression Master Mix, according to manufacturer'sprotocol. Ratio between HN mRNA verses F mRNA was calculated using Δct.

Examination of MuV neurotoxicity. The rat neurotoxicity test wasperformed as described before (Rubin et al., 2000, J Virol;74:5382-5384). Newborn rats were inoculated intracerebrally with 100 pfuof rMuV (n=36), or rMuVΔSH (n=24) in 20 μl. EMEM. Animals weresacrificed at one month after injection and the brains were removed,immersion fixed and embedded in paraffin. One 10 μm sagittal section ata constant distance from the anatomical midline from each hemisphere ofbrain was selected, and stained with haematoxylin and eosin. Theneurotoxicity score was calculated based on the cross-sectional area ofthe brain (excluding the cerebellum) as a percentage of the lateralventricle on tissue sections from paired brain using Image-Pro Plusimage analysis software (Media Cybernetics). The neurotoxicity score wasdefined as the mean ratio (percentage) of these two measurements on eachof the two tissue sections per rat brain. Any rats with signs of pain ordistress prior to the planned 1 month end point were humanely euthanizedimmediately and included in analyses. The NIH Guidelines for the Careand Use of Laboratory Animals were strictly adhered to throughout.

The results of this example can now also be found in Xu et al., “Rescueof wild-type mumps virus from a strain associated with recent outbreakshelps to define the role of the SH ORF in the pathogenesis of mumpsvirus,” Virology; 417(1):126-36 (published Aug. 15, 2011; Epub 2011 Jun.14).

Example 2 The V Protein of Mumps Virus Plays a Critical Role inPathogenesis

Mumps virus (MuV) causes an acute infection in humans characterized by awide array of symptoms ranging from relatively mild manifestations, suchas parotitis, to more-severe complications, such as meningitis andencephalitis. Widespread mumps vaccination has reduced mumps incidencedramatically; however, outbreaks still occur in vaccinated populations.The V protein of MuV, when expressed in cell culture, blocks interferon(IFN) expression and signaling and interleukin-6 (IL-6) signaling. Inthis example, a recombinant MuV incapable of expressing the V protein(rMuVΔV) was generated. The rescued MuV was derived from a clinicalwild-type isolate from a recent outbreak in the United States(MuV^(Iowa/US/06), G genotype). Analysis of the virus confirmed theroles of V protein in blocking IFN expression and signaling and IL-6signaling. It was also found that the rMuV^(Iowa/US/06)ΔV virus inducedhigh levels of IL-6 expression in vitro, suggesting that V plays a rolein reducing IL-6 expression. In vivo, the rMuV^(Iowa/US/06)ΔV virus washighly attenuated, indicating that the V protein plays an essential rolein viral virulence.

The RNA genome of MuV is 15,384 nucleotides long. It encodes nine knownviral proteins. The V protein of MuV has 224 amino acid residues andcontains a cysteine (Cys)-rich C terminus that is conserved among allparamyxoviruses. The V protein interrupts the interferon (IFN) signalingpathway through degradation of STAT1, a critical transcription factorfor IFN-activated gene expression (Kubota et al., 2002, J Virol;76:12676-12682). A tryptophan-rich motif within the Cys-rich C terminusof the MuV V protein is essential in the ubiquitination and degradationof STAT1 (Kubota et al., 2002, J Virol; 76:12676-12682; Kubota et al.al., 2001, Biochem Biophys Res Commun; 283:255-259; and Nishio et al.,2002, Virology; 300:92) through the N-terminal region of STAT1 (Yokosawaet al., 2002, J Virol; 76:12683-12690). The V protein has also beendemonstrated to associate with receptor-activated C kinase (RACK1),which contains Trp-Asp (WD) repeats and mediates interactions betweenthe IFN receptor and STAT1. The V-RACK1 interaction results in thedisassociation of STAT1 and RACK1, contributing to the blockade of IFNsignaling by V protein (Kubota et al., 2002, J Virol; 76:12676-12682).This interaction may be important to block IFN signaling before thecomplete degradation of STAT1 occurs (Kubota et al., 2005, J Virol;79:4451-4459). The V protein of MuV also interacts with MDA5, a RNAhelicase that plays a critical role in the activation of IFN expressionin infected cells (Andrejeva et al., 2004, Proc Natl Acad Sci USA;101:17264-17269) and blocks the activation of IFN expression. TheCys-rich C terminus of V protein is essential for its interaction withMDA5 through its helicase C domain (Parisien et al., 2009, J Virol;83:7252-7260; Ramachandran and Horvath, 2010, J Virol; 84:11152-11163).The V protein can serve as a substrate for inhibitor of κB kinase ε(IKKe)/tumor necrosis factor receptor associated factor (TRAF) familymember-associated NF-κB activator (TANK)-binding kinase 1 (TBK1),resulting in inhibition of the activation of interferon regulatoryfactor 3 (IRF3). The interaction between V protein and TBK1/IKKeinhibits the activation of IRF3, a critical transcription factor for IFNexpression, resulting in the blockade of IFN expression (Lu et al.,2008, J Biol Chem; 283:14269-14276). The V protein causes degradation ofSTAT3, a critical transcription factor for interleukin-6 (IL-6)-mediatedsignaling and oncogenesis (Ulane et al., 2003, J Virol; 77:6385-6393). Apoint mutation within the V protein (E to D at position 95) results in aV protein that is capable of STAT1 degradation without affecting itsability to target STAT3 for degradation. The ability of V protein toblock IFN signaling is thought to be important for viral pathogenesis(Rosas-Murrieta et al., 2010, Virol J; 7:263). In this Example arecombinant MuV that it was no longer capable of expressing the Vprotein (rMuV^(Iowa/US/06)ΔV) was generated. The rescued MuV was derivedfrom a clinical wild-type (WT) isolate from a recent outbreak in theUnited States (MuV^(Iowa/US/06), G genotype). This is the first study ofthe functions of the V protein of MuV in the context of viral infection.

Materials and Methods

Plasmids, viruses, and cells. The MuV strain, MuV^(Iowa/US/06), wasobtained from a patient during the 2006 Midwest mumps outbreak in theUnited States. A full-length cDNA clone of the virus (pMuV^(Iowa/US/06))was constructed as described in Example 1 (see also Xu et al., 2011,Virology; 417:126-136). This plasmid was modified to not express the Vprotein by changing the editing site of the P/V gene (GGGGGG;nucleotides 1-6 of SEQ ID NO:14) to GAGGAGGG (nucleotides 1-8 of SEQ IDNO:15) and the addition of another four base pairs (CTAG; nucleotides3-6 of SEQ ID NO:16) to the 3′ untranslated region (3′ UTR; SEQ IDNO:16) of the gene to comply with “the rule of six” (Kolakofsky et al.,1998, J Virol; 72:891-899).

To rescue an infectious virus, plasmid pMuV^(Iowa/US/06) (5 μg), alongwith plasmids pCAGGS-L (1 μg), pCAGGS-NP (1.5 μg), and pCAGGS-P (200ng), were transfected into BSRT-7 cells. Three days later, transfectedBSRT-7 cells were mixed with Vero cells at 1:1. Ten to 14 days later,when syncytium formation was observed, supernatants containingrMuV^(Iowa/US/06)ΔV were collected and plaque purified in Vero cells.Plaques (developing 4 to 7 days postinfection [dpi]) were amplified inVero cells, and their genomes were sequenced. The rescue procedure wasrepeated to produce independent stocks of rMuV^(Iowa/US/06)ΔV.

Vero, HeLa, MDBK, and L929 cells were maintained in Dulbecco's modifiedEagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1%penicillin-streptomycin (P/S) (Mediatech Inc., Holu Hill, Fla.). BSRT-7cells were maintained in DMEM supplemented with 10% FBS, 1% P/S, and 10%tryptose phosphate broth (TPB), plus 400 μg/ml Geneticin G418antibiotic. Cells were cultured at 37° C. with 5% CO₂ and passaged theday before infection or transfection at appropriate dilution factors toachieve 80 to 90% confluence the next day. For virus infection, cellswere inoculated with viruses in DMEM plus 1% bovine serum albumin (BSA)at a multiplicity of infection (MOI) of 0.01, 3, or 5 and incubated for1 to 2 h at 37° C. with 5% CO₂. The inocula were then replaced with DMEMsupplemented with 2% FBS and 1% P/S. Cells were transfected withplasmids using PLUS and Lipofectamine reagents (Invitrogen, Carlsbad,Calif.) following the manufacturer-provided protocols.

All mumps viruses were grown in Vero cells and were harvested at 4 to 7dpi. Virus titers were measured in Vero cells by plaque assay asdescribed previously (He and Lamb, 1999, J Virol; 73:6228-6234; He etal., 1997, Virology; 237:249-260). Parainfluenza virus 5 (PIV5) andrecombinant PIV5 lacking the expression of the C terminus of the Vprotein (rPIV5VΔC) were grown as described before (He et al., 2002,Virology; 303:15-32).

Sequencing of viruses. Viral RNA was extracted from cell culturesupernatants by using the QIAamp viral RNA extraction minikit (QiagenInc., Valencia, Calif.) following manufacturer's protocol. Isolatedviral RNA was reverse transcribed into cDNA by using SuperScript IIIreverse transcriptase with random hexamers (Invitrogen). SynthesizedcDNA then served as templates for PCR using mumps virus genome-specificprimers (shown in Table 1) and Taq polymerase (Invitrogen). Fifteen setsof primers (shown in Table 2), each containing a forward and reverseprimer, were designed to divide the genome into 15 overlappingfragments. The primers were then used for the subsequent sequencing ofthe PCR products (Li et al., 2006, Virology; 346:219-228). Leader andtrailer sequences were sequenced following the standard protocol ofrapid amplification of cDNA ends (RACE) (Li et al, 2011, J Virol;85:32-42).

Flow cytometry and TUNEL assay. Flow cytometry was performed aspreviously described (36). HeLa or Vero cells in 6-well plates were mockinfected or infected with rMuV^(Iowa/US/06)ΔV, rMuV^(Iowa/US/06), orMuV^(Iowa/US/06) at an MOI of 0.1 or 0.5. At 24 h postinfection (hpi),48 hpi, 72 hpi, or 96 hpi, attached cells were trypsinized and combinedwith floating cells in the culture media. Cells were centrifuged andresuspended in 0.5% paraformaldehyde in phosphate-buffered saline (PBS)for 1 h at 4° C. The fixed cells were then washed with PBS andpermeabilized in 50% fetal calf serum (FCS)-50% DMEM plus three volumesof 70% ethanol overnight. Permeabilized cells were subjected to eitherterminal deoxynucleotidyltransferase-mediated dUTP-biotin nick endlabeling (TUNEL) staining or MuV^(Iowa/US/06)-NP, MuV^(Iowa/US/06)-P, orMuV^(Iowa/US/06)-HN staining for protein expression level. For NPstaining, monoclonal MuV^(Iowa/US/06)-NP antibody was diluted 1:200; forP staining, monoclonal MuV^(Iowa/US/06)-P antibody (as described inExample 1; see also Xu et al., 2011, Virology; 417:126-136) was diluted1:50 followed by fluorescein isothiocyanate (FITC) anti-mouse secondaryantibody (Jackson ImmunoResearch) staining at a dilution of 1:10,000.For HN staining, polyclonal MuV^(Iowa/US/06)-HN was diluted 1:50followed by FITC anti-rabbit secondary antibody staining at a dilutionfactor of 1:10,000. TUNEL staining was performed as described beforefollowing the manufacturer's protocol (Roche) (Sun et al., 2009, PLoSPathog; 5:e1000525; Sun et al., 2004, J Virol; 78:5068-5078).

Immunoblotting. Vero cells in 6-well plates at approximately 90%confluence were mock infected or infected with rMuV^(Iowa/US/06) orrMuV^(Iowa/US/06)ΔV at an MOI of 0.01 or 0.5. Cells were lysed andcollected at different time points postinfection in 0.5 ml WCEB buffer(50 mM Tris-HCl [pH 8.0], 120 mMNaCl, 0.5% NP-40, 0.00076% EGTA, 0.2 mMEDTA, 10% glycerol) with a mixture of protease inhibitors as describedpreviously (Rubin et al., 2011, Vaccine; 29:2850-2855; Rubin et al.,2000, J Virol; 74:5382-5384). Cell lysates were briefly centrifuged toremove cell debris and loaded onto a 10% or 17.5% polyacrylamide gel andsubjected to SDS-PAGE. Proteins were transferred to an Immobilon-FLtransfer membrane (Millipore, Billerica, Mass.), incubated with primaryantibody (anti-MuV^(Iowa/US/06) V, 1:500; anti-MuV^(Iowa/US/06) NP,1:5,000; anti-MuV^(Iowa/US/06) P, 1:2,000 [43], anti-STAT1, 1:200(#B2410; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.);anti-STAT2, 1:200 (#07-224; Millipore, Billerica, Mass.); anti-STAT3,1:200 (#F300; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) andcorresponding secondary antibodies conjugated to horseradish peroxidase,and detected using an Amersham ECL Western blotting detection kit (GEHealthcare Bioscience, Piscataway, N.J.).

Growth curve of rMuV^(Iowa/US/06)ΔV and rMuV^(Iowa/US/06). Cells in 6-cmplates or 6-well plates were infected with rMuVΔV or rMuV at an MOI of0.01. One milliliter (6-cm plates) or 100 μl (6-well plates) ofsupernatant were collected at 0 h, 24 h, 48 h, and 72 h (24 h, 48 h, 72h, 120 h, 168 h, 216 h, and 264 h in HeLa) postinfection, supplementedwith 1% BSA, and stored at 80° C. Virus titers were determined by plaqueassay using Vero cells in 6-well plates in triplicate. After one to twohour (h) incubations with the viruses, the growth medium was changed toDMEM with 2% FBS, 1% P/S, and 1% low-melting-point agarose. Four to 7dpi, 6-well plates of Vero cells were stained with Giemsa stain, andplaques were counted.

ELISA for IFN-β and IL-6. HeLa cells or 293T cells were mock infected orinfected with PIV5-WT (MOI 5), rPIV5-VΔC (MOI-5), rMuV^(Iowa/US/06) (MOI0.5), or rMuV^(Iowa/US/06)ΔV (MOI 0.5) virus in 12-well plates. Thesupernatants were collected at 24 h and 48 h postinfection. The amountof secreted IL-6 in the medium was measured using the OptEIA human IL-6enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Jose,Calif.), and IFN-β was measured using the VeriKine human IFN-β ELISA kitas described before (16, 18) (PBL InterferonSource, Piscataway, N.J.)according to the manufacturer's instructions.

Neurotoxicity test. The neurovirulence phenotype of the rescued viruseswas assessed by measuring the extent of MuV-induced hydrocephalus, themajor neuropathologic outcome of MuV infection in rats, as previouslydescribed (Rubin et al., 2000, J Virol; 74:5382-5384). Briefly, threelitters of 8 to 10 newborn Lewis rats were inoculated intracerebrallywith 10 μl of DMEM containing 100 PFU of each of the two virus stocksrescued from plasmid pMuV^(Iowa/US/06) and each of the two virus stocksrescued from plasmid pMuV^(Iowa/US/06)ΔV. On day 30 postinoculation, therats were humanely sacrificed by CO₂ asphyxiation following the NIHGuidelines for the Care and Use of Laboratory Animals. Brains wereremoved and immersion fixed in 10% neutral-buffered formalin at 4° C.for 4 to 5 days, followed by paraffin embedding. Sagittal sectionsobtained at a standard distance from either side of the rostral-caudalmidline were stained with hematoxylin and eosin.

The neurovirulence score was determined by calculating the ratio betweenthe cross-sectional area of the brain (excluding the cerebellum) and thecross-sectional area of the lateral ventricle (which is enlargedfollowing infection with neurovirulent MuV strains), measured usingImage Pro Plus image analysis software (Media Cybernetics, SilverSpring, Md.). The mean ratio (given in percent) of these twomeasurements on each of the two tissue sections per rat brain is theneurovirulence score for that particular brain. The neurovirulence scorefor each virus is the mean neurovirulence score for all brains withinthe treatment group. All comparisons were made using at test or, withnonnormal data (failed Shapiro-Wilk test), the Mann-Whitney rank sumtest (α=0.05).

Results

Recovery of a recombinant MuV lacking expression of V protein (rMuVΔV).To investigate the role of the V protein in viral pathogenesis in thecontext of viral infection, we constructed a cDNA of theMuV^(Iowa/US/06) genome containing mutations to ablate the V proteinexpression (pMuV^(Iowa/US/06)ΔV) (the accession number forMuV^(Iowa/US/06) genome is JN012242) (Xu et al., 2011, Virology;417:126-136). Ablation of the V protein expression from the genome wasachieved by changing the editing site (GGGGGG; nucleotides 1-6 of SEQ IDNO:14) in the P/V gene into GAGGAGGG (nucleotides 1-8 of SEQ ID NO:15).Therefore, only a transcript encoding the P protein is generated fromP/V gene transcription (FIG. 9A). Infectious viruses abolishing theexpression of the V protein (rMuV^(Iowa/US/06)ΔV) were rescued from thecloned DNA through transfection of pMuV^(Iowa/US/06)ΔV into BSRT-7cells. Rescued viruses were further plaque purified and amplified inVero cells. To confirm the presence of the genetic changes to shut offthe V protein expression in the rescued virus genome, viral RNAs wereextracted from virus stocks and reverse transcribed into cDNA forsequencing (FIGS. 9B and 9C).

Sequencing of the genome of the rescued virus revealed the presence ofnucleotide substitutions in the NP gene end (GE) sequence and at the P/Vgene start (GS) sequence comparing to input cDNA sequence as well as thechanges that would ablate the expression of the V protein (FIG. 9C).Immunoblotting of infected cells was performed to confirm the absence ofthe V protein expression in rMuV^(Iowa/US/06)ΔV-infected Vero cells(FIG. 9D). To further investigate, the virus was rescued from the cDNAplasmid seven more times (FIG. 10A). Viruses from seven out of the totaleight rescued viruses contained a point mutation at either the NP GE(six) or P/V GS region (one), while one contained a point mutation inthe L gene (FIG. 10B). All of the rescued rMuV^(Iowa/US/06)ΔV virusescontained at least one point mutation in their genome, and the mostfrequent point mutation was at position 1899 in the genome; thus, thisvirus was used as a representative virus and designated asrMuV^(Iowa/US/06)ΔV for this work, unless otherwise noted.

Analysis of rMuVΔV in tissue culture cell lines. To analyze the growthrate of rMuV^(Iowa/US/06)ΔV in cell lines, Vero cells or HeLa cells wereinfected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of0.01, medium was collected at multiple time points postinfection, andviral titers were determined using plaque assay (FIG. 11A).rMuV^(Iowa/US/06)ΔV grew at a rate comparable to that ofrMuV^(Iowa/US/06)V in Vero cells during the first 48 h postinfection(hpi), and then the growth of rMuV^(Iowa/US/06)ΔV decreased and remainedapproximately 1 log lower in titer than rMuV^(Iowa/US/06) throughout thestudied time course (FIG. 11A). Plaque size of rMuV^(Iowa/US/06)ΔV inVero cells showed no significant differences from that ofrMuV^(Iowa/US/06) (FIG. 11B). Protein expression levels ofrMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) low-MOI-infected Vero cellswere examined by immunoblotting with anti-NP, P, and V or anti-β-actin(FIG. 11C). Consistent with the time course, the viral proteinexpression levels of rMuV^(Iowa/US/06)ΔV were similar to those ofrMuV^(Iowa/US/06) at 48, 72, and 96 hpi (adjusting for the levels ofβ-actin). In HeLa cells, the growth of rMuV^(Iowa/US/06)ΔV was reduced(FIG. 11D). The absence of a functional V protein reduced the virustiter of rMuV^(Iowa/US/06)ΔV by almost 2 log₁₀ from 72 hpi to 168 hpicompared with rMuV^(Iowa/US/06). Nevertheless, the both viruses reachedsimilar titers at later time points.

Expression of viral genes in rMuV^(Iowa/US/06)ΔV-infected cells.Mutations at either the NP GE or P/V GS in recovered rMuV^(Iowa/US/06)ΔVviruses suggested that a modulation of viral protein expression levelsbetween NP and P might be critical for the recovery ofrMuV^(Iowa/US/06)ΔV from cDNA. To investigate the viral proteinexpression pattern in rMuV^(Iowa/US/06)ΔV, Vero cells infected with ahigh MOI were stained for NP and P proteins at different time pointspostinfection and assessed by flow cytometry (FIG. 12). To quantify thepossible changes in the NP and P expression pattern, P proteinexpression levels were normalized to that of the corresponding NP levels(FIGS. 12A and 12B). The P/NP ratio of rMuVΔV was significantly higherthan that of rMuV at 12, 16, 24, and 48 hpi, indicating an elevated Pprotein expression in the rMuV^(Iowa/US/06)ΔV virus. This difference wasno longer evident by 72 hpi.

To investigate if this altered NP and P expression pattern was uniquefor this rMuV^(Iowa/US/06)ΔV strain, an rMuV^(Iowa/US/06)ΔV containing aP GS mutation (rMuV^(Iowa/US/06)ΔV [P GS]) and an rMuV^(Iowa/US/06)ΔVcontaining a L open reading frame (ORF) mutation (rMuV^(Iowa/US/06)ΔV [Lgene]) were also examined (FIGS. 12C and 12D). Similar torMuV^(Iowa/US/06)ΔV, both rMuV^(Iowa/US/06)ΔV (PGS) andrMuV^(Iowa/US/06)ΔV (L gene) had a P protein expression level greaterthan that of rMuV^(Iowa/US/06), suggesting that this altered NP and Pexpression pattern was typical for the recovered rMuV^(Iowa/US/06)ΔVviruses.

To examine if downstream viral protein expression was affected by eitherdeletion of the V protein or the point mutation in NP GE, HN expressionlevels were examined using flow cytometry. Vero cells were either mockinfected or infected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at anMOI of 0.5, and then cells were collected at 24 hpi and subjected to NPand HN staining (FIG. 13). No significant changes in the HN-to-NP ratiowere observed.

rMuV^(Iowa/US/06)ΔV-induced accelerated CPE in tissue culture celllines. rMuV^(Iowa/US/06)ΔV-induced cytopathic effects (CPE) werecompared in three different cell culture lines from three differentorganisms. HeLa (human), Vero (monkey), and MDBK (bovine) cells wereinfected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06) at an MOI of 0.5,and the cells were photographed at 72 hpi. rMuV^(Iowa/US/06)ΔV causedthe most-severe CPE in HeLa cells. More and larger syncytia wereobserved in rMuV^(Iowa/US/06)ΔV-infected HeLa cells, which may be amajor contributing factor to cell death (FIG. 14A). To examine whetherthe cell death was caused by apoptosis, the TUNEL assay was performed(FIG. 14B). HeLa cells infected with rMuV^(Iowa/US/06)ΔV at an MOI of0.5 showed at least a 2-fold higher level of apoptosis than cellsinfected with rMuV. Similarly, rMuV^(Iowa/US/06)ΔV induced a higherlevel of apoptosis in Vero cells (FIG. 14C). That the lack of V led toan increase in apoptosis in infected cells suggests that the V proteinmight play a role in blocking induction of apoptosis in infected cells.

Status of STAT proteins in MuV^(Iowa/US/06)-infected cells. Previousstudies have shown that the V protein is involved in blocking the IFNsignaling pathway by targeting STAT proteins for degradation. Todetermine whether MuV^(Iowa/US/06)V protein is the only virus-encodingantagonist of the IFN pathway, STAT family protein levels were examinedin Vero cells infected with rMuV^(Iowa/US/06)ΔV or rMuV^(Iowa/US/06)(FIGS. 15A and 15B). Consistent with previous in vitro transfectionstudies, STAT1 and STAT3, but not STAT2, were completely degraded inrMuV^(Iowa/US/06)-infected Vero cells, while rMuV^(Iowa/US/06)ΔV, whichlacks expression of the V protein, failed to target any STAT proteinsfor degradation, indicating that the V protein might be essential andnecessary for STAT protein degradation by MuV^(Iowa/US/06). There is atime lag between the occurrence of a detectable V protein and thedegradation of STAT protein (FIGS. 15A and 15B), implying that thedegradation of STATs might require accumulation of the V protein.

PIV5, a paramyxovirus closely related to MuV, prevents induction ofIFN-β in infected cells, while recombinant PIV5 lacking the expressionof the conserved C terminus of the V protein does not (He et al., 2002,Virology; 303:15-32; Poole et al., 2002, Virology; 303:33-46). Tocompare IFN-β inductions by rMuV^(Iowa/US/06)ΔV and rMuV^(Iowa/US/06),IFN-β concentration in the medium of infected 293T cells was measured byusing ELISA. At 48 hpi, rMuV^(Iowa/US/06)ΔV induced IFN-β productionhigher than that induced by rMuV^(Iowa/US/06) (FIG. 16A), indicatingthat the V protein of MuV^(Iowa/US/06) plays a role in limiting IFN-βexpression.

rMuV^(Iowa/US/06)ΔV led to a higher level of IL-6 induction. Toinvestigate whether the absence of a functional V protein inMuV^(Iowa/US/06) infection would lead to induction of other cytokines,IL-6 production levels in the medium of rMuV^(Iowa/US/06)ΔV andrMuV^(Iowa/US/06)-infected cells were examined. At 48 hpi,rMuV^(Iowa/US/06)ΔV led to a higher level of IL-6 production thanrMuV^(Iowa/US/06) in HeLa cells (FIG. 16B), indicating that IL-6induction was reduced by the presence of the V protein. Intriguingly,rMuV^(Iowa/US/06) infection also induced a significant amount of IL-6production. This is consistent with MuV being an inflammatory disease.

Neurotoxicity of rMuV^(Iowa/US/06)ΔV. To examine the effect of the Vprotein on virus neurovirulence, viruses from two independent rescuesusing plasmid pMuV^(Iowa/US/06)ΔV (rMuV^(Iowa/US/06)ΔV) (FIG. 10A) weretested in rats, along with rMuV^(Iowa/US/06) and the highly attenuatedJeryl Lynn (JL) vaccine virus as controls. As shown in FIG. 17, the ΔVviruses were highly attenuated compared to rMuV^(Iowa/US/06) and JLvaccine virus.

Discussion

The results of this example confirm these findings through the study ofa recombinant virus derived from a clinical isolate (genotype G)ablating the expression the V protein in the context of in vitroinfection.

The lack of V protein expression also led to the induction of a higherlevel of IL-6, a proinflammatory cytokine, suggesting that the V proteinplays a role in suppressing IL-6 expression. The lack of V proteinexpression in infected cells likely resulted in the attenuation of thisstrain in an animal model, suggesting that the V protein plays anessential role in viral virulence. It is possible that the inability ofrMuV^(Iowa/US/06)ΔV to counter IFN action limited the replication of thevirus in vivo, and the induction of a higher level of IL-6 byrMuV^(Iowa/US/06)ΔV attracted monocytes to clear the infection quickly,resulting in the attenuation of rMuV^(Iowa/US/06)ΔV in vivo.

Genetically, the closest virus to MuV is parainfluenza virus 5 (PIV5).The V proteins of MuV and PIV5 share many identical functions, includingblocking IFN expression through MDA5, blocking IFN signaling throughdegradation of STAT1, and inhibiting expression of IL-6 invirus-infected cells. Interestingly, a recombinant PIV5 lacking theentire V protein has never been obtained in tissue culture cells,suggesting that the V protein of PIV5 plays a more critical role invirus replication (Dillon and Parks, 2007, J Virol 81:11116-11127; He etal., 2002, Virology; 303:15-32) than the V protein does for MuV. Theviability of rMuV^(Iowa/US/06)ΔV suggests that the role ofMuV^(Iowa/US/06) V protein in virus replication is dispensable, at leastin tissue culture cells.

In this example, a recombinant virus incapable of producing the Vprotein (rMuV^(Iowa/US/06)ΔV) was generated using a reverse geneticssystem for MuV based on a clinical isolate from a recent outbreak. Thisvirus grew to titers similar to those for wild-type virus in Vero cells,a cell line that is used for vaccine production, as well as in othercell types. Most importantly, the virus exhibited low neurotoxicity inrats, supporting it as a vaccine candidate.

The V/P gene of MuV encodes three proteins, V, I, and P, through aprocess of “RNA editing,” in which nontemplate G residues are insertedinto mRNA during transcription at a specific site to generate mRNAs thatcan be translated into three different ORFs (Saito et al., 1996,Microbiol Immunol; 40:271-275). The V protein is translated from the“unedited” copy of mRNA, P from the mRNA with two G residue insertions,and the I protein from the mRNA with one or four G residue insertions.All of these proteins have identical N termini of 155 amino acidresidues. The P protein has 391 amino acid residues and plays anessential role in viral RNA synthesis. The I protein has 170 amino acidresidues, and its function is unclear. It is possible that the I mRNA isa by-product of RNA editing and it may not have any significantfunctions. The strategy we used to generate rMuV^(Iowa/US/06)ΔV alsoeliminated expression of the I protein. Because the mRNA for 1 countsonly for less than 2% of total V, I, and P transcripts, and its sequenceis very similar to the N termini of V and P (I has about 170 amino acidresidues and 155 of them are identical to the N termini of V and P)(Paterson and Lamb, 1990, J Virol; 64:4137-4145; Takeuchi et al., 1990,Virology; 178:247-253), the phenotypes of rMuV^(Iowa/US/06)ΔV isattributed to the lack of V protein. However, a possible role for the Iprotein cannot be excluded.

All changes except the one in the L gene occurred in the gene junctionbetween NP and P/P genes to generate viable infectious MuV incapable ofexpressing the V protein. It is interesting that a mutation in the Lgene was able to allow the rescue of a virus lacking the V protein.While the possibility that the mutation in the L gene occurredfortuitously cannot be excluded and is immaterial to the function of L,one can speculate that the particular mutation may play a role inmodulating interactions between NP-P and L, considering that all otherviruses rescued had mutations to modulate the levels of NP and P.Further analysis of the virus may lead to a better understanding of thefunction of L.

The results of this example can now also be found in Xu et al., “The vprotein of mumps virus plays a critical role in pathogenesis,” J Virol;86(3):1768-76 (February 2012; Epub 2011 Nov. 16).

Example 3 Immunogenicity of MuVΔSH and MuVΔV in Mice

The immunogenicity of rMuVΔSH and rMuVΔV in mice was determined andMuV-specific immune responses measured. Mice in a group of 10 wereinoculated with PBS, or 10⁶ pfu of MuV, rMuVΔV or rMuVΔSH intranasally.At 21 days post inoculation, blood samples from the mice were collected.Titers of anti-MuV antibodies in the sera were measured using ELISA. The96-well plates for ELISA were coated with purified MuV virion. P valuesfor MuV and rMuVΔSH, MuV and MuVΔV at highest dilution and lowestdilution of sera were lower than 0.05. The results are shown in FIG. 18.

Further humoral immunity (antibody) analysis will include adetermination of anti-MuV antibodies in bronchoalveolar lavage (BAL), asmeasured by MuV-specific ELISA. In ELISA assays, the isotypes (IgA,IgG1, IgG2a, IgG2b, and IgG3) of the antibodies will also be determinedusing appropriate secondary antibodies. MuV-specific antibody titerswill also be measured by virus. Neutralization assays againstheterogonous JL or homologous MuV-IA will be performed on serum and BALwash samples.

Cell mediated immunity (T cell) may be measured by antigen-specific IFNγproduction. Specifically, lymphocytes from the BAL, spleen and/ordraining lymph node will be assayed for MuV-specific T cell responses byrestimulation with MuV-infected APCs or with purified MuV virions thatare disrupted with mild detergent. IFN-γ responses will be determined byintracellular cytokine staining and/or ELISPOT assays.

Since the site of induction of immune responses can alter the nature ofthe immune response and dramatically impact protective efficacy, localand systemic immunity to MuV will be measured at various time pointsafter immunization. Intranasal (IN) MuV immunization has the potentialto induce local MuV-specific T cell and immunoglobulin responses thatmediate protection against MuV challenge. Local (i.e. lung) MuV-specificimmune responses will be assessed by analysis of BAL samples collectedat time points after immunization or challenge. Infiltrating lymphocytepopulations will be collected by centrifugation and the bronchoalveolarlavage (BAL) will be analyzed for mucosal Ig. Systemic responses will beassessed by analysis of serum antibody and splenic or mediastinal lymphnode (MLN) lymphocytes.

Current MMR vaccination regimen calls for two-dose intramuscular (IM)inoculation. A similar regimen was used in a mouse model to evaluateefficacies of vaccines (Cusi et al., 2001, Arch Virol; 146(7):1241-862).Initially, immunogenicity may be assayed using such a two dose/IMregiment. The mice will be injected with a primary dose and followed byan injection at two weeks after initial injection. At one month afterlast immunization, the mice will be sacrificed for immunological assaysas described above. This experiment will generate a baseline of immuneresponses after inoculation with the vaccine candidates, along with theJL vaccine in our hand.

The immunogenicity of a rMuV vaccine construct as described herein maybe examined using a two-dose/intranasal (IN) protocol. Both humoral andcell-mediated immune responses will be measured. It has been reportedthat the IN route generated better immune responses for some vaccines,including a robust cell mediated immune responses. In addition, INinoculation has the benefit of generating mucosal immune responses andhigher titers of IgA. Because of the success of using the IN route forinfluenza virus vaccination, the IN route will be feasible route for thenew vaccine to be introduced to a large human population.

In a similar fashion, a three-dose inoculation regimen will also betested. As the most likely target for initial Phase I clinical trialswill be healthy individual who have already been vaccinated withtwo-dose MMR, the immune responses after two dose/IM inoculation withthe JL vaccine followed by a third dose of JL (as a control) or a thirddose of MuV vaccine either by IM or IN will be examined. If a MuVvaccine construct as described herein used as a boost (third dose) issafe and generates robust anti-genotype G immune responses, it may beused to replace the second dose of MMR and may eventually replace JL inthe two-dose MMR.

The immunogenicity of any of the rMuV constructs described herein may beassayed in mice, as described in this example. Similar immunogenicityand efficacy studies may also be undertaken in additional animal modelsystems, including, but not limited to, ferret and non-human primatemodel systems.

Example 4 Generation and Analysis of rMuVΔSHΔV

Because MuV vaccine is used in 1-year old infants, safety is a paramountconsideration in developing a new vaccine. While both rMuVΔSH and rMuVΔVdemonstrate attenuation in a rat brain-based neurotoxicity test, tofurther reduce any potential risk, a recombinant virus lacking both SHand V (rMuVΔSHΔV) was generated using the reverse genetics system.

Briefly, following protocols described in more detail in Example 1 (see,for example, FIG. 3A), the ablation of SH protein expression from theMuVΔSHΔV genome was achieved by deleting 156 nucleotides in the SH geneopen reading frame (ORF) of the SH gene from pMuV-IA. And, followingprotocols described in more detail in Example 2 (see, for example, FIG.9A), the ablation of the V protein expression from the MuVΔSHΔV genomewas achieved by changing the editing site (GGGGGG) in the P/V gene intoGAGGAGGG. Therefore, only a transcript encoding the P protein isgenerated from P/V gene transcription. Infectious viruses abolishing theexpression of both the SH protein and the V protein (rMuVΔSHΔV) wererescued from the cloned DNA through transfection of pMuVΔSHΔV intoBSRT-7 cells. Rescued viruses were further plaque purified and amplifiedin Vero cells. To confirm the presence of the genetic changes to shutoff both SH and V protein expression in the rescued virus rMuVΔSHΔVgenome, viral RNAs were extracted from virus stocks, reverse transcribedinto cDNA, and sequenced.

Following procedures described in more detail in Example 1 and Example2, immunoblotting of infected cells will be performed to confirm theabsence of SH and V protein expression in rMuVΔSHΔV-infected cells. Theexpression, function, immunogenicity, and pathogenicity of rMuVΔSHΔVwill be analyzed by a variety of methods, including, but not limited to,any of those described herein, for example, as described in the Examplesincluded herewith. Studies may include examination of the neurotoxicityof rMuVΔSHΔV in the neonatal rat brain and examination of immunogenicityin mice.

Example 5 Improving Recombinant MuV as a Vaccine Candidate

In this example, rMuV constructs will be further mutated using thereverse genetics system to introduce mutations at desirable locations.The resultant MuV mutants will be analyzed in tissue culture cells.Neurotoxicity will be evaluated in a rat model and immunogenicity of theviruses examined in mice, ferrets, and primates. It is likely that rMuVlacking the V and SH plus additional point mutations will be the mostattenuated and the least likely to be reverted.

Generation and analysis of additional MuV mutants. The closest virus toMuV is parainfluenza virus 5 (PIV5). These two viruses have identicalnumber of genes and gene order. In recent studies of PIV5, residueswithin PIV5 proteins have been identified that are capable of enhancingviral gene expression and inducing expression of cytokines such as typeI interferon (Sun et al., 2009, PLoS Pathog; 5(7):e1000525). It wasfound that the residue of S157 of the P protein of PIV5 is a bindingsite for host kinase PLK1 and the residue of 5308 of the P protein ofPIV5 is a phosphorylation site of PLK1. Mutating S157 or 5308 to aminoacid residue A, results in a virus that increases viral gene expressionas well as induction of interferon-β expression. Increasing viral geneexpression will potentially increase immune responses because ofincreased amount of antigens and increasing IFN expression will likelycause attenuation because of anti-viral effects of IFN. Correspondingresidues within the P protein of MuV are T147 and S307. These residueswill be mutated and the impact of changing these residues on viral geneexpression and induction of interferon will be examined.

Generation and analysis of rMuV lacking I and rMuVΔV expressing I. Thestrategy used in Example 2 to generate rMuVΔV also eliminated expressionof the I protein besides the expression of V. The I protein is anediting product of VI//P gene. Its function is not known. Because itsexpression level is very low compare with V or P, and its sequence isvery similar to the N-terminal of V and P (I has about 170 amino acidresidues and 155 of them are identical to the N-terminal of V and P),the effect of deleting the I protein has often been overlooked. For thepurposes of developing an effective vaccine, deleting I along with V maybe advantageous for attenuation. However, it is possible that the Iprotein does have a role in viral pathogenesis and contributes toefficacy of a vaccine. To investigate the role of the I protein in viruslife cycle in general, and in generating immune responses in particular,a recombinant virus lacking the I protein will be generated. The rMuVΔVgenome will be used as a backbone to insert V between P and M. As aresult of the mutations at the editing site, no I or V will be made fromthe P gene, but the V protein will be made from the newly inserted V.Similarly, the I gene will be inserted between HN and L in the backboneof the rMuVΔV genome to generate a recombinant rMuVΔV expressing I(rMuVΔ V+I). The reason for two different gene junctions to be insertedis that the V protein expression level ought to be high to reflect thewild type virus infection and expression level of I should be low as inwild type virus infected cells. Gene junction closer to the leadersequence (P-M junction) will give higher viral gene expression levelsthan the distant one (HN-L junction). The resultant viral construct willbe analyzed as described in the previous examples.

Generation and analysis of revertants. In the case of rMuVΔV, severalpoint mutations were introduced into the genome of MuV to give rise tothe V protein deletion phenotype. It is possible that mutations thatwill revert the phenotype may be generated over a period of time. Whilea revertant of rMuVΔV has not been obtained after passing the virus inVero cells over 20 passages, this experiment will be repeated ininterferon competent cell lines. Vero cells are WHO and FDA-approved forvaccine production and do not produce type I IFN. That rMuVΔV has beenstable in this cell line is encouraging for future mass production ofrMuVΔV as a vaccine. However, Vero cells are defective in IFN productiondue to a deletion of IFN gene locus. Thus, the rate of revertant ofrMuVΔV in an interferon competent environment will be examined. A549cells, a human lung cell line that produces and responses tointerferons, will be infected with rMuVΔV at a MOI of 0.1 and at 4 dayspost infection, media of the infected cells will be collected and usedto infect fresh A549 cells at about 0.1 MOI. In preliminary studies, itwas observed that rMuVΔV reached about 10⁶ pfu/ml and this titer will beused as a rough estimation for our experiment. Virus will be collectedat every passage from the media of rMuVΔV-infected A549 cells andinitially sequence viruses from passage 5, 10, 15 and 20. Similarly,other MuV mutants such as rMuVΔSH and rMuV-P-T147A will be examined.

Recombinant viruses that demonstrate enhanced viral gene expressionand/or increased interferon induction will be tested for neurotoxicityand immunogenicity, as described in the previous example. Even mutationsthat do not achieve attenuation equal to rMuVΔV will be tested, becauseof their potential in induction of type I interferon. While Type Iinterferon is well known for its anti-viral activities, it also plays apositive role in inducing adaptive immunity (Iwasaki et al., 2004, NatImmunol; 5(10):987-95). It promotes proliferation of memory T cells andprevents apoptosis of T cell. It plays a critical role in antigen crosspresentation. It enhances humoral immunity and stimulates dendriticcells. It will be of significant interest if these IFN inducing MuVmutants generate more robust immune responses than its parent. If thesemutations indeed produce better immune responses, these mutations willbe incorporated into the rMuV genome.

It is possible that the I mRNA is a by-product of RNA editing and it maynot have any significant functions. Investigating whether the I proteinhas a role in virus replication and pathogenesis will not only revealpotential novel functions about the I protein, it will also be importantfor vaccine development. In case rMuVΔV is too attenuated, expressing Iin the backbone of rMuVΔV may help to design a virus with desirablelevel of attenuation. In the case of human PIV2 vaccine development,deleting the V protein resulting in a virus that is too attenuated to beeffective (Schaap-Nutt et al., 2010, Virology; 397(2):285-98). Thus,adding V or I back may be result in a more appropriately attenuated MuVvaccine.

It is possible that the residues in the P protein of MuV that areresponsible for PLK1 binding and phosphorylation may be different thanpredicted above. They will be searched using an approach similar to thatused for PIV5: there are two PLK1 binding motifs within the P of MuV.Analogous residues in MuV will be examined. In preliminary studies, ithas been found that the P protein and PLK1 interacted, indicating that aPLK1 binding site is within the P protein. In addition, mutations withinP have been identified that enhanced its ability to facilitate viralgene expression, i.e., increased viral gene expression phenotype.Besides mutating the P protein, mutations will also be made in othergenes. For instance, mutations in the L gene of PIV5 that enhances viralgene expression have been identified. The same mutations will beincorporated into the L gene of MuV.

Example 6 Immunogenicity of Recombinant Mumps Viruses in Ferrets

The immunogenicity and efficacy as a vaccine candidate of any of the MuVdescribed herein will be tested in ferrets. The ferret is a small animalmodel system for the study of the pathogenesis of MuV infection. Thereis a remarkable similarity in the lung physiology and morphology betweenferrets and humans. Ferrets are highly susceptible to infection withrespiratory viruses. Ferrets have been established as an animal modelfor several other respiratory pathogens. Most importantly, MuV has beenisolated from infected ferrets and pathological changes were observed inthe lungs of infected animals (Gordon et al., 1956, J Immunol;76(4):328-33). Studies may include the infection of ferrets with a rMuVconstruct, the determination of immunogenicity of a rMuV construct inferrets, and an examination of the efficacies of a such vaccinecandidate in reducing virus load and pathological changes in lungs afterchallenge. As previously described (Gordon et al., 1956, J Immunol;76(4):328-33), ferrets in a group of 5 will be infected with 10⁷ pfu ofwild type MuV or a rMuV construct in 1 ml volume. Animals will bemonitored for fever daily in the first week and every other day insecond week after infection. At 3, 4, 5, 7, 9 and 11 days afterinoculation, nasal washes and blood samples will be collected and titersof virus in them will be determined using plaque assay. At 3, 4, 7, 11and 14 days after inoculation, ferrets will be sacrificed and lungs andturbinates will be collected and titers of virus will be determined.Pathological changes in lungs and turbinates will be examined using H&Estaining. The immunogenicity of MuV mutants in ferrets will be examined,as described in the previous examples. Humoral immunity and cellularimmunity against MuV will be examined after inoculation with the vaccinecandidates as well as the JL vaccine and wild type MuV. Two-dose INinoculation and three-dose (two-dose IM inoculation with JL followed bya single IN inoculation of the vaccine candidates) may be used. Besidesimmunological tests, vaccinated animals will be challenged with wildtype MuV. Reagents for immunological assays in ferrets, includingreagents for assaying cellular immune responses, will be generated. Suchreagents may include monoclonal antibodies against CD3, CD4, CD8, IFN-β,IFN-γ, IL-6, and IL-8.

Example 7 Mumps Virus as a Vector for Respiratory Syncytial VirusVaccine Development

Respiratory syncytial virus (RSV) is the most important cause ofpediatric viral respiratory infection and is a major cause of morbidityand mortality among infants as well as immunocompromised subjects andthe elderly (Collins, P. L., R. M. Chanock, and B. R. Murphy,Respiratory syncytial virus, in Fields Virology, D. M. Knipe and P. M.Howley, Editors. 2001, Lippincott, Williams and Wilkins: Philadelphia.p. 1443-1485.). In addition, severe RSV infection can result in wheezingand asthma later in life. Unlike infection by other respiratory viruses,RSV does not induce long-lasting protective immunity against subsequentinfection. Thus, most individuals are infected multiple times throughoutthe course of their lives. Currently, there is no vaccine for RSV, norare there effective curative treatments for severe RSV disease althoughaerosolized ribavirin and prophylactic immunoglobulin therapy are usedin the clinical setting. However, the high cost of palivizumabprophylaxis raises the question of cost-effectiveness relative to healthbenefits due to the need for monthly injections during RSV season.Therefore, there is a pressing need for safe and effective vaccine forRSV.

As a negative non-segmented single-stranded RNA virus (NNSV), MuV is agood viral vector candidate for vaccine development because it does nothave a DNA (or nuclear) phase in its life cycle, and thus the possibleunintended consequences of genetic modifications of host cell DNAthrough recombination or insertion are avoided. In comparison topositive strand RNA viruses, the genome structure of MuV is stable.Thus, MuV is better suited as a vaccine vector than positive strand RNAviruses since the genomes of positive strand RNA viruses recombine andoften delete the inserted foreign genes quickly.

Generation and analysis of MuV-F containing RSV F. The F gene of RSV (A2strain) will be inserted between F and SH of MuV genome using the samestrategy as for the generation of MuV-GFP (MuV-F). Briefly, the F genewill be combined with the gene end (GE), intergenic region (I) and genestart (GS) (which are important for viral mRNA synthesis), using afour-primer PCR approach (He et al., 1995, Gene; 164:75-79). Thesequences will be inserted between GS of NP and the coding sequence ofthe NP gene. Although the “rule of six”, which viral RNA genome requiresto be multiple of six to be effective, is not absolute for MuV, thelength of the genome with F will be maintained to be a multiple of six.Expression levels of F in MuV-F-infected cells will be examined usingimmunoprecipitation in comparison to RSV-infected cells. Growth rates ofthe virus at high and low MOI will be compared to MuV.

Generation and examination of MuV-F containing 3′-proximal F as avaccine candidate. Negative strand RNA viruses, such as MuV, initiatetranscription from the 3′ end leader sequence, and transcription levelsof the viral genes are affected by their distances to the leadersequence. For example, the NP gene of MuV, which is the closest to theleader sequence, is the most abundantly transcribed, whereas the L genethat is the located most distant from the leader sequence is leasttranscribed (FIG. 19). It is expected that the efficacy of the vaccinecandidate will be enhanced by increasing the expression level of the Fprotein (as has been shown for recombinant RSV (Krempl et al., 2002, JVirol; 76(23):11931-42)). To increase the expression level of the Fgene, the F gene will be inserted immediately downstream of the leadersequence and upstream of the NP gene (F-MuV) (FIG. 19).

The RSV G protein can be similarly expressed as the RSV F protein usingmumps virus as a vector.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO:1 Mumps virus genome including V/P gene encoding a V    protein and SH gene encoding a small hydrophobic protein-   SEQ ID NO:2 Mumps virus V protein-   SEQ ID NO:3 Mumps virus small hydrophobic protein-   SEQ ID NO:4 SH protein sequence for Mumps virus strain Glouc1/UK96-   SEQ ID NO:5 SH protein sequence for Mumps virus strain UK01-22-   SEQ ID NO:6 SH protein sequence for Mumps virus strain MuV-IA-   SEQ ID NO:7 nucleic acid sequence upstream of SH gene-   SEQ ID NO:8 recombinant nucleic acid sequence resulting from    deletion of SH gene-   SEQ ID NO:9 PCR product sequenced to confirm deletion of SH protein-   SEQ ID NO:10 MuV-IA SH N-terminal peptide sequence used to generate    antibody-   SEQ ID NO:11 MuV-IA SH C-terminal peptide sequence used to generate    antibody-   SEQ ID NOs:12-13 MuV-IA V protein peptide sequence used to generate    antibody-   SEQ ID NO:14 nucleic acid editing sequence within the P/V gene-   SEQ ID NO:15 recombinant nucleic acid sequence eliminating V protein    expression-   SEQ ID NO:16 recombinant nucleic acid sequence resulting from    modification of P/V gene-   SEQ ID NO:17 PCR product sequenced to confirm deletion of V protein-   SEQ ID NO:18 nucleic acid sequence from the end of the NP gene to    the start of the P/V gene a Mumps virus having a V protein deletion-   SEQ ID NO:19-25 rescued nucleic acid sequence from the end of the NP    gene to the start of the P/V gene in a Mumps virus having a V    protein deletion-   SEQ ID NO:26-81 synthetic oligonucleotide primer

1. An isolated nucleotide sequence comprising the cDNA sequence encodingthe full length RNA genome of a mumps virus, wherein the isolatednucleotide sequence encodes a mumps virus unable to express a smallhydrophobic (SH) protein product and/or express a V protein product. 2.The isolated nucleotide sequence comprising the cDNA sequence encodingthe full length RNA genome of a mumps virus of claim 1 comprising adeletion of the open reading frame (ORF) encoding the SH protein, amutation converting a start codon into a stop codon, or a mutation inthe region between the ORF encoding the F polypeptide and the ORFencoding the SH polypeptide that disrupts transcription of the SH gene.3. The isolated nucleotide sequence comprising the cDNA sequenceencoding the full length RNA genome of a mumps virus of claim 2comprising a deletion of the ORF encoding the SH protein.
 4. Theisolated nucleotide sequence comprising the cDNA sequence encoding thefull length RNA genome of a mumps virus of claim 3 comprising a deletionof 156 nucleotides of the ORF encoding the SH protein.
 5. The isolatednucleotide sequence comprising the cDNA sequence encoding the fulllength RNA genome of a mumps virus of claim 1 comprising one or moremutations to the V/I/P gene abrogating expression of the V protein. 6.The isolated nucleotide sequence comprising the cDNA sequence encodingthe full length RNA genome of a mumps virus of claim 5, wherein the oneor more mutations to the V/I/P gene abrogating expression of the Vprotein comprise the nucleotide sequence GAGGAGGG at the editing site inthe P/V gene.
 7. The isolated nucleotide sequence comprising the cDNAsequence encoding the full length RNA genome of a mumps virus of claim 1comprising: a deletion of the open reading frame (ORF) encoding the SHprotein, a mutation converting a start codon into a stop codon, or amutation in the region between the ORF encoding the F polypeptide andthe ORF encoding the SH polypeptide that disrupts transcription of theSH gene; and one or more mutations to the V/I/P gene abrogatingexpression of the V protein.
 8. The isolated nucleotide sequencecomprising the cDNA sequence encoding the full length RNA genome of amumps virus of claim 7 wherein the one or more mutations to the V/I/Pgene abrogating expression of the V protein comprise the nucleotidesequence GAGGAGGG at the editing site in the P/V gene.
 9. The isolatednucleotide sequence comprising the cDNA sequence encoding the fulllength RNA genome of a mumps virus of claim 1 comprising a furthermutation and/or deletion.
 10. The isolated nucleotide sequencecomprising the cDNA sequence encoding the full length RNA genome of amumps virus of claim 9 comprising a mutation or deletion effectingphosphorylation of the P protein, a mutation at T147 and/or a mutationat S307 of the P protein.
 11. (canceled)
 12. The isolated nucleotidesequence comprising the cDNA sequence encoding the full length RNAgenome of a mumps virus of claim 1 further comprising a mutation of theL gene.
 13. The isolated nucleotide sequence comprising the cDNAsequence encoding the full length RNA genome of a mumps virus of claim 1further comprising expression of an I protein product.
 14. The isolatednucleotide sequence comprising the cDNA sequence encoding the fulllength RNA genome of a mumps virus of claim 1, wherein the mumps genomefurther encodes a heterologous polypeptide.
 15. (canceled)
 16. Theisolated nucleotide sequence comprising the cDNA sequence encoding thefull length RNA genome of a mumps virus of claim 1 wherein the mumpsvirus is MuV/IowaUS/2006 (MuV-IA).
 17. An isolated nucleotide sequencecomprising the cDNA sequence encoding the full length RNA genome of theMUV/IowaUS/2006 (MuV-IA) strain of the mumps virus.
 18. The isolatednucleotide sequence of claim 17, wherein the nucleotide sequencecomprises SEQ ID NO:1.
 19. A recombinant mumps virus (rMuV) or plasmidencoding a measles virus genome (pMuV) comprising an isolated nucleotideacid sequence of claim
 1. 20. A plasmid comprising the isolatednucleotide sequence of claim 1, or a fragment thereof. 21-26. (canceled)27. A method of inducing an immune response to mumps virus in a subject,the method comprising administering an effective amount of an isolatednucleotide sequence plasmid, pMuV, rMuV, viral particle, or compositionof claim 1 to the subject.
 28. A method of vaccinating a subject againstmumps, the method comprising administering an effective amount of anisolated nucleotide sequence plasmid, pMuV, rMuV, viral particle, orcomposition of claim 1 to the subject. 29-30. (canceled)